Methods and compositions for inhibiting cd32b expressing cells in igg4-related diseases

ABSTRACT

The present disclosure relates to immunoglobulins that bind FcγRIIb+ B cells and coengage CD19 on the cell&#39;s surface and an FcγRIIb on the cell&#39;s surface, methods for their generation, and methods for using the immunoglobulins for the treatment of an IgG4-related disease.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit under 35 U.S.C. §119 of: U.S.Provisional Patent Application No. 62/347,419, entitled “METHODS ANDCOMPOSITIONS FOR INHIBITING CD32B EXPRESSING CELLS,” filed on Jun. 8,2016; U.S. Provisional Patent Application No. 62/399,896, entitled“METHODS AND COMPOSITIONS FOR INHIBITING CD32B EXPRESSING CELLS INIGG4-RELATED DISEASES,” filed on Sep. 26, 2016; and U.S. ProvisionalPatent Application No. 62/421,261, entitled “METHODS AND COMPOSITIONSFOR INHIBITING CD32B EXPRESSING CELLS IN IGG4-RELATED DISEASES,” filedon Nov. 12, 2016.

SEQUENCE LISTING

A Sequence Listing submitted with this disclosure is hereby incorporatedby reference.

TECHNICAL FIELD

The present disclosure relates to immunoglobulins that bind FcγRIIb+ Bcells and coengage CD19 on the cell's surface and an FcγRIIb on thecell's surface, methods for their generation, and methods for using theimmunoglobulins for the treatment of an IgG4-related disease.

BACKGROUND

Antigen recognition by B cells is mediated by the B cell receptor (BCR),a surface-bound immunoglobulin in complex with signaling componentsCD79a (Igα) and CD79b (Igβ). Crosslinking of BCR upon engagement ofantigen results in phosphorylation of immunoreceptor tyrosine-basedactivation motifs (ITAMs) within CD79a and CD79b, initiating a cascadeof intracellular signaling events that recruit downstream molecules tothe membrane and stimulate calcium mobilization. This leads to theinduction of diverse B cell responses (e.g., cell survival,proliferation, antibody production, antigen presentation,differentiation, etc.) which lead to a humoral immune response(DeFranco, A. L., 1997, Curr. Opin. Immunol. 9, 296-308; Pierce, S. K.,2002, Nat. Rev. Immunol. 2, 96-105; Ravetch, J. V. & Lanier, L. L.,2000, Science 290, 84-89). Other components of the BCR coreceptorcomplex enhance (e.g., CD19, CD21, and CD81) or suppress (e.g., CD22 andCD72) BCR activation signals (Doody, G. M. et al., 1996, Curr. Opin.Immunol. 8, 378-382; Li, D. H. et al., 2006, J. Immunol. 176,5321-5328). In this way, the immune system maintains multiple BCRregulatory mechanisms to ensure that B cell responses are tightlycontrolled.

When antibodies are produced to an antigen, the circulating level ofimmune complexes (e.g., antigen bound to antibody) increases. Theseimmune complexes downregulate antigen-induced B cell activation. It isbelieved that these immune complexes downregulate antigen-induced B cellactivation by coengaging cognate BCR with the low-affinity inhibitoryreceptor FcγRIIb, the only IgG receptor on B cells (Heyman, B., 2003,Immunol. Lett. 88, 157-161). It is also believed that this negativefeedback of antibody production requires interaction of the antibody Fcdomain with FcγRIIb since immune complexes containing F(ab′)₂ antibodyfragments are not inhibitory (Chan, P. L. & Sinclair, N. R., 1973,Immunology 24, 289-301). The intracellular immunoreceptor tyrosine-basedinhibitory motif (ITIM) of FcγRIIb is necessary to inhibit BCR-inducedintracellular signals (Amigorena, S. et al., 1992, Science 256,1808-1812; Muta, T., et al., 1994, Nature 368, 70-73). This inhibitoryeffect occurs through phosphorylation of the FcγRIIb ITIM, whichrecruits SH2-containing inositol polyphosphate 5-phosphatase (SHIP) toneutralize ITAM-induced intracellular calcium mobilization (Kiener, P.A., et al., 1997, J. Biol. Chem. 272, 3838-3844; Ono, M., et al., 1996,Nature 383, 263-266; Ravetch, J. V. & Lanier, L. L., 2000, Science 290,84-89). In addition, FcγRIIb-mediated SHIP phosphorylation inhibits thedownstream Ras-MAPK proliferation pathway (Tridandapani, S. et al.,1998, Immunol. 35, 1135-1146).

SUMMARY OF EXEMPLARY EMBODIMENTS

The present disclosure provides methods of using an immunoglobulin toinhibit cells that express FcγRIIb. The FcγRIIb⁺ cell inhibitory methodsdisclosed herein comprise treating an IgG4-related disease (IgG4-RD) ina patient. The method comprises administering an immunoglobulin thatbinds FcγRIIb and CD19 on the surface of a B cell, wherein saidimmunoglobulin comprises an Fc region, wherein said Fc region is an Fcvariant of a parent Fc polypeptide, comprising at least one amino acidsubstitution selected from the group consisting of 234W, 235I, 235Y,235R, 235D, 236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y,wherein numbering is according to the EU index as in Kabat. In someembodiments, the at least one substitution may be selected from thegroup consisting of 267E and 328F. In other embodiments, the Fc variantmay comprise at least two amino acid substitutions in the Fc regioncompared to the parent Fc polypeptide, wherein said at least twosubstitutions is selected from the group consisting of 235D/267E,235Y/267E, 235D/S267D, 235I/267E, 235I/267D, 235Y/267D, 236D/267E,236D/267D, 267E/328F, 267D/328F, 268D/267E, 268D/267D, 268E/267E, and268E/267D. In other embodiments, the at least two substitutions may be267E/328F. In other embodiments, the Fc region that binds FcγRIIbcomprises SEQ ID NO:7 and SEQ ID NO:9.

Also disclosed herein, is a method of reducing at least one symptomassociated with IgG4-RD in a subject. The method may compriseadministering an immunoglobulin that binds FcγRIIb and CD19 on thesurface of a B cell, wherein said immunoglobulin comprises an Fc region,wherein said Fc region is an Fc variant of a parent Fc polypeptide,comprising at least one amino acid substitution selected from the groupconsisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D,267E, 268E, 268D, 328F, and 328Y, wherein numbering is according to theEU index as in Kabat. In some embodiments, the at least one substitutionmay be selected from the group consisting of 267E and 328F. In otherembodiments, the Fc variant may comprise at least two amino acidsubstitutions in the Fc region compared to the parent Fc polypeptide,wherein said at least two substitutions is selected from the groupconsisting of 235D/267E, 235Y/267E, 235D/S267D, 235I/267E, 235I/267D,235Y/267D, 236D/267E, 236D/267D, 267E/328F, 267D/328F, 268D/267E,268D/267D, 268E/267E, and 268E/267D. In other embodiments, the at leasttwo substitutions may be 267E/328F. In other embodiments, the Fc regionthat binds FcγRIIb comprises SEQ ID NO:7 and SEQ ID NO:9.

In some embodiments, the at least one symptom associated with IgG4-RDmay be reduced within 7 days of administration of the immunoglobulin. Infurther embodiments, that at least one symptom associated with IgG4-RDis reduced within 14 days of administration of the immunoglobulin. Inother embodiments, the at least one symptom is exhibited in an organselected from lymph nodes, submandibular glands, parotid glands,lacrimal glands, kidney, heart, pericardium, orbit, nasal cavity, lungs,bile ducts, salivary glands, and pancreas.

Also disclosed herein, is a method of depleting plasmablasts in asubject with an IgG4-related disease. The method may compriseadministering an immunoglobulin that binds FcγRIIb and CD19 on thesurface of a B cell, wherein said immunoglobulin comprises an Fc region,wherein said Fc region is an Fc variant of a parent Fc polypeptide,comprising at least one amino acid substitution selected from the groupconsisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D,267E, 268E, 268D, 328F, and 328Y, wherein numbering is according to theEU index as in Kabat. In some embodiments, the at least one substitutionmay be selected from the group consisting of 267E and 328F. In otherembodiments, the Fc variant may comprise at least two amino acidsubstitutions in the Fc region compared to the parent Fc polypeptide,wherein said at least two substitutions is selected from the groupconsisting of 235D/267E, 235Y/267E, 235D/S267D, 235I/267E, 235I/267D,235Y/267D, 236D/267E, 236D/267D, 267E/328F, 267D/328F, 268D/267E,268D/267D, 268E/267E, and 268E/267D. In other embodiments, the at leasttwo substitutions may be 267E/328F. In other embodiments, the Fc regionthat binds FcγRIIb comprises SEQ ID NO:7 and SEQ ID NO:9.

In some embodiments, the depletion of plasmablasts is observed within 7days following administration the immunoglobulin that binds FcγRIIb andCD19 on the surface of a B cell. In other embodiments, the plasmablastsmay be depleted by at least 10% relative the number of plasmablastsprior to the administration of immunoglobulin. In other embodiments, theplasmablasts may be depleted by at least 20% relative the number ofplasmablasts prior to the administration of immunoglobulin. In otherembodiments, the plasmablasts may be depleted by at least 30% relativeto baseline. In other embodiments, the plasmablasts may be depleted byat least 40% relative the number of plasmablasts prior to theadministration of immunoglobulin. In other embodiments, the plasmablastsmay be depleted by at least 80% relative the number of plasmablastsprior to the administration of immunoglobulin.

Additionally disclosed herein, is a method of reducing CD4+SLAMF7+CTLcell number in a subject with an IgG4-related disease. The method maycomprise administering an immunoglobulin that binds FcγRIIb and CD19 onthe surface of a B cell, wherein said immunoglobulin comprises an Fcregion, wherein said Fc region is an Fc variant of a parent Fcpolypeptide, comprising at least one amino acid substitution selectedfrom the group consisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N,239D, 267D, 267E, 268E, 268D, 328F, and 328Y, wherein numbering isaccording to the EU index as in Kabat. In some embodiments, the at leastone substitution may be selected from the group consisting of 267E and328F. In other embodiments, the Fc variant may comprise at least twoamino acid substitutions in the Fc region compared to the parent Fcpolypeptide, wherein said at least two substitutions is selected fromthe group consisting of 235D/267E, 235Y/267E, 235D/S267D, 235I/267E,235I/267D, 235Y/267D, 236D/267E, 236D/267D, 267E/328F, 267D/328F,268D/267E, 268D/267D, 268E/267E, and 268E/267D. In other embodiments,the at least two substitutions may be 267E/328F. In other embodiments,the Fc region that binds FcγRIIb comprises SEQ ID NO:7 and SEQ ID NO:9.

In some embodiments, the reduction of CD4+SLAMF7+CTL cell number may beobserved within 24 days following administration the immunoglobulin thatbinds Fc RIIb and CD19 on the surface of a B cell. In other embodiments,the CD4+SLAMF7+CTL cells are reduced by at least 10% relative the numberof CD4+SLAMF7+CTL cells prior to the administration of immunoglobulin.

Disclosed herein, is a method of treating a disease in a subject. Themethod may comprise administering an immunoglobulin that binds Fc RIIband CD19 on the surface of a B cell, wherein said immunoglobulincomprises an Fc region, wherein said Fc region is an Fc variant of aparent Fc polypeptide, comprising at least one amino acid substitutionselected from the group consisting of 234W, 235I, 235Y, 235R, 235D,236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y, whereinnumbering is according to the EU index as in Kabat, and wherein thedisease is selected from the group consisting of IgG4-relatedsialadenitis (chronic sclerosing sialadenitis, Küttner's tumour,Mikulicz's disease), IgG4-related dacryoadenitis (Mikulicz's disease),IgG4-related ophthalmic disease (idiopathic orbital inflammatorydisease, orbital pseudotumor), chronic sinusitis, eosinophilicangiocentric fibrosis, IgG4-related hypophysitis (IgG4-relatedpanhypophysitis, IgG4-related adenohypophysitis, gG4-relatedinfundibuloneurohypophysitis, autoimmune hypophysitis), IgG4-relatedpachymeningitis, IgG4-related leptomeningitis (idiopathic hypertrophicpachymeningitis), IgG4-related pancreatitis (Type 1 autoimmunepancreatitis, IgG4-related AIP, lymphoplasmacytic sclerosingpancreatitis, chronic pancreatitis with diffuse irregular narrowing ofthe main pancreatic duct), IgG4-related lung disease (Pulmonaryinflammatory pseudotumour), IgG4-related pleuritis, IgG4-relatedhepatopathy, IgG4-related sclerosing cholangitis, IgG4-relatedcholecystitis, IgG4-related aortitis (inflammatory aortic aneurysm),IgG4-related periaortitis (chronic periaortitis), IgG4-relatedperiarteritis, IgG4-related pericarditis, IgG4-related mediastinitis(fibrosing mediastinitis), IgG4-related retroperitoneal fibrosis(retroperitoneal fibrosis, Albarran-Ormond syndrome, Ormond's disease(tetroperitoneal fibrosis)), perirenal fasciitis, Gerota'sfasciitis/syndrome, periureteritis fibrosa, sclerosing lipogranuloma,sclerosing retroperitoneal granuloma, non-specific retroperitonealinflammation, sclerosing retroperitonitis, retroperitoneal vasculitiswith perivascular fibrosis), IgG4-related mesenteritis (subtypes are:mesenteric panniculitis, mesenteric lipodystrophy and retractilemesenteritis) (sclerosing mesenteritis, systemic nodular panniculitis,liposclerosis mesenteritis, mesenteric Weber-Christian disease,mesenteric lipogranuloma, xanthogranulomatous mesenteritis),IgG4-related mastitis (sclerosing mastitis), IgG4-related kidney disease(IgG4-RKD), IgG4-related tubulointerstitial nephritis (IgG4-TIN),IgG4-related membranous glomerulonephritis (idiopathictubulointerstitial nephritis), IgG4-related prostatitis, IgG4-relatedperivasal fibrosis (chronic orchialgia), IgG4-related paratesticularpseudotumor, IgG4-related epididymo-orchitis (paratesticular fibrouspseudotumor, inflammatory pseudotumor of the spermatic cord,pseudosarcomatous myofibroblastic proliferations of the spermatic cord,proliferative funiculitis, chronic proliferative periorchitis,fibromatous periorchitis, nodular periorchitis, reactive periorchitis,fibrous mesothelioma), IgG4-related lymphadenopathy, IgG4-related skindisease (angiolymphoid hyperplasia with eosinophilia, cutaneouspseudolymphoma), IgG4-related perineural disease, and IgG4-relatedthyroid disease (Reidel's thyroiditis), eosinophilic angiocentricfibrosis (affecting the orbits and upper respiratory tract),inflammatory pseudotumour, and multifocal fibrosclerosis. In someembodiments, the disease may be selected from the group consisting ofautoimmune pancreatitis (lymphoplasmacytic scleorising pancreatitis),eosinophilic angiocentric fibrosis (affecting the orbits and upperrespiratory tract), fibrosing mediastinitis, idiopathic hypertrophicpachymeningitis, idiopathic tubulointerstitial nephritis, inflammatorypseudotumour, Küttner's tumour, Mikulicz's disease, fibrosclerosis,periaortitis, periarteritis, inflammatory aortic multifocal aneurysm,Ormond's disease (tetroperitoneal fibrosis), Riedel's thyroiditis, andsclerosing mesenteritis. In some embodiments, the at least onesubstitution may be selected from the group consisting of 267E and 328F.In other embodiments, the Fc variant may comprise at least two aminoacid substitutions in the Fc region compared to the parent Fcpolypeptide, wherein said at least two substitutions is selected fromthe group consisting of 235D/267E, 235Y/267E, 235D/S267D, 235I/267E,235I/267D, 235Y/267D, 236D/267E, 236D/267D, 267E/328F, 267D/328F,268D/267E, 268D/267D, 268E/267E, and 268E/267D. In other embodiments,the at least two substitutions may be 267E/328F. In other embodiments,the Fc region that binds FcγRIIb comprises SEQ ID NO:7 and SEQ ID NO:9.

Administration of an immunoglobulin to a subject by any of the methodsdisclosed herein, may also comprise a reduction of the subject's IgG4-RDresponder index score (IgG4-RD RI score). In some embodiments, within 2weeks following administration of the immunoglobulin, the subject'sIgG4-RD RI score is reduced by at least 1 from the baseline score. Inother embodiments, the IgG4-RD RI score is reduced by 3 within 2 weeksfollowing administration of the immunoglobulin.

Administration of an immunoglobulin to a subject by any of the methodsdisclosed herein, may also comprise administering about 1 to about 10mg/kg body weight of the immunoglobulin to the subject. In someembodiments, about 5 mg/kg body weight of the immunoglobulin isadministered to the subject. In some embodiments, the immunoglobulin isadministered to the subject every 14 days. In other embodiments, theimmunoglobulin is administered to the subject every 14 days for at least2 doses. In other embodiments, the immunoglobulin is administered to thesubject every 14 days for at least 6 doses. In other embodiments, theimmunoglobulin is administered to the subject every 14 days for at least12 doses.

Administration of an immunoglobulin to a subject by any of the methodsdisclosed herein, may further comprise administering standard treatmentsfor an IgG4-related disease including anti-inflammatory pain relieverdrugs (NSAIDs such as aspirin, ibuprofen, naproxen, or Celebrex),acetaminophen, steroids, glucocorticoids (i.e. prednisone),immunosuppressive agents (i.e. azathioprine, mycophenolate mofetil), andimmunosuppressive biologics (i.e. rituximab, bortezomib). In furtherembodiments, any of the methods disclosed herein may further comprisetapering and/or discontinuing the use of steroids. In other embodiments,the subject of any of the methods disclosed herein may be relapsed orrelapsed or refractory to rituximab.

The B cell of any of the methods disclosed herein, may be selected fromthe group consisting of a plasma cell and a plasmablast. In furtherembodiments, B cell may be a plasmablast.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Alignment of the amino acid sequences of the human IgGimmunoglobulins IgG1, IgG2, IgG3, and IgG4. FIG. 1A provides thesequences of the CH1 (Cγ1) and hinge domains, and FIG. 1B provides thesequences of the CH2 (Cγ2) and CH3 (Cγ3) domains. Positions are numberedaccording to the EU index of the IgG1 sequence, and differences betweenIgG1 and the other immunoglobulins IgG2, IgG3, and IgG4 are shown ingray. Allotypic polymorphisms exist at a number of positions, and thusslight differences between the presented sequences and sequences in theprior art may exist. The possible beginnings of the Fc region arelabeled, defined herein as either EU position 226 or 230.

FIGS. 2A-2B. Common haplotypes of the human gamma1 (FIG. 2A) and gamma2(FIG. 2B) chains.

FIG. 3. Methods of inhibiting B cell activation. Here CR represents aco-receptor of the BCR complex, but could be any antigen expressed onany FcγRIIb+ cell.

FIG. 4. Biacore surface plasmon resonance sensorgrams showing binding ofFc variant anti-CD19 antibodies to human FcγRIIb.

FIG. 5A-5D. Affinities of Fc variant antibodies for human FcγRs asdetermined by Biacore surface plasmon resonance. The graph shows the−log(KD) for binding of anti-CD19 variant and WT IgG1 antibodies tohuman FcγRI (I), R131 FcγRIIa (RIIa), H131 FcγRIIa (HIIa), FcγRIIb(IIb), and V158 FcγRIIIa (Villa). Binding of L235Y/S267E, G236D/S267E,and S267E/L328F to V158 FcγRIIIa was not detectable.

FIGS. 6A-6F. Binding of Fc variant antibodies to human FcγRs relative toWT IgG1 as measured by cell surface binding. Antibodies (variant and WTIgG1) were added to HEK293T cells transfected with FcγRIIb to assesscell surface binding. The binding curves were constructed by plottingMFI as a function of Fc variant concentration.

FIG. 7. Affinities of Fc variant antibodies for human FcγRs asdetermined by Biacore surface plasmon resonance. The graph shows the−log(KD) for binding of anti-CD19 variant and WT IgG1 antibodies tohuman FcγRI (I), R131 FcγRIIa (RIIa), H131 FcγRIIa (HIIa), FcγRIIb(lib), and V158 FcγRIIIa (Villa).

FIGS. 8A-8B. ATP-dependent B cell viability assay demonstrating thesurvival of primary human B cells upon BCR activation, here carried outby crosslinking with anti-mu (FIG. 8A) or anti-CD79b (FIG. 8B)antibodies.

FIG. 9. Inhibition of B cell proliferation by Fc variant anti-CD19antibodies. Anti-RSV (Respiratory Syncytial Virus) S267E/L328F is usedas a control (RSV is not expressed on B cells). An ATP-dependentluminescence assay was used to measure B cell proliferation in thepresence of 10 μg/ml anti-CD79b activating antibody, and the effect ofanti-CD19-S267E/L328F was compared to anti-CD19-IgG1 (native IgG1 Fvcontrol) and anti-RSV-S267E/L328F (non-CD19 Fc control). To assess theimportance of CD19 and FcγRIIb coengagement, anti-RSV-S267E/L328F aloneor in combination with anti-CD19-IgG1 was used.

FIG. 10. Anti-CD19-S267E/L328F inhibits the anti-apoptotic effects ofBCR activation on primary human B cells. Inhibition of BCR-mediatedsurvival signals by FcγRIIb and CD19 coengagement was examined usingannexin-V staining in the presence of 10 μg/mi anti-CD79b. B cellapoptosis was stimulated by anti-CD19-S267E/L328F, but notanti-CD19-IgG1 (Fv control), anti-RSV-S267E/L328F (Fc control), or thetwo controls combined.

FIGS. 11A-11B. Evaluation of the capacity of co-engagement of CD19 andFcγRIIb to inhibit human B cell activation in vivo. (FIG. 11A) Schematicrepresentation of the experimental protocol. (FIG. 11B) Titer ofanti-tetanus toxoid (TT) specific antibody in huPBL-SCID mice after TTimmunization and treatment with vehicle (PBS), anti-CD19 IgG1 WT,anti-CD19 with enhanced FcγRIIb affinity (a-CD19 S267E/L328F), oranti-CD20 (Rituximab).

FIG. 12. ATP-dependent luminescence assay measuring B cell proliferationin the presence of 1 μg/ml anti-CD79b-SN8-G236R/L328R antibody, andvarying concentrations of either enhanced FcγRIIb variant (S267E/L328F)or FcγR knockout variant (G236R/L328R or ̂236R/L328R) versions ofanti-CD20 (clone PRO70769), -CD52 (Campath), and -CD19 (HuAM4G7)antibodies.

FIG. 13. ATP-dependent luminescence assay measuring B cell proliferationin the presence of 1 μg/ml anti-CD79b-SN8-G236R/L328R antibody, andvarying concentrations of either enhanced FcγRIIb variant (S267E/L328F)or FcγR knockout variant (G236R/L328R or ̂236R/L328R) versions ofanti-CD19 antibodies (clones HD37, 21D4, or HuAM4G7.

FIGS. 14A-14D. FIG. 14A lists the amino acid sequences of variousvariable regions, heavy chain constant regions, and full lengthantibodies. FIG. 14B is a continuation of the list in FIG. 14A. FIG. 14Cis a continuation of the list in FIG. 14A and FIG. 14B. FIG. 14D is acontinuation of the list in FIGS. 14A to 14C.

FIGS. 15A-15B. FIG. 15A is a schematic of an anti-CD19 antibodyS267E/L328F, an immunoglobulin that comprises an Fc domain that bindsFcγRIIb and an Fv domain that binds CD19. FIG. 15B is a schematicshowing that the anti-CD19 antibody S267E/L328F binds to FcγRIIb+ cellsand coengages CD19 thereby enhancing the natural regulatory role ofFcγRIIb (inhibition of B cell activation).

FIG. 16. Number of organs involved at baseline. Median number was 4(range 1 to 10).

FIG. 17. Active organs at baseline. Organ site involvement occurring ata frequency of 50% included lymph nodes, submandibular glands, parotidglands, and lacrimal glands.

FIG. 18. IgG4-RD responder index over time. Twelve of fifteen patients(80%) have had an initial response to an anti-CD19 antibody withmodifications S267E/L328F of a 2 point reduction in the IgG4-RD RIwithin 2 weeks of the first dose. Five patients have achieved an IgG4-RDRI of 0 (no disease activity).

FIGS. 19A-19C. FIG. 19A demonstrates that administration of 2 mg/kg ofan anti-CD19 antibody S267E/L328F results in an inhibition of thestimulated expression of CD86 that then slowly returned to baseline atabout 100 days following administration of the anti-CD19 antibodyS267E/L328F. FIG. 19B demonstrates that administration of 2 mg/kg of theanti-CD19 antibody S267E/L328F results in a reversible decline inperipheral B cell counts which return to normal at about 40-60 daysfollowing administration of the anti-CD19 antibody S267E/L328F. FIG. 19Cdemonstrates that subjects challenged with tetanus antigen and thenadministered varying doses of the anti-CD19 antibody S267E/L328F rangingfrom 0.03 to 10 mg/kg exhibited a detectable drop in anti-tetanus IgGrelative to placebo-treated subjects.

FIGS. 20A-20G. Flow cytometry gating strategy for B cells andplasmablasts.

FIG. 21. Summary of B cell numbers during treatment. Percentage ofinitial total B cell (CD79b+) number is shown for fourteen patients.Each line represents an individual patient (fourteen patientsrepresented).

FIGS. 22A-22B. FIG. 22A shows the effect of antibody treatment on CD79+plasmablast (CD79b+CD3-CD30-CD27hiCD38hi) number. FIG. 22B shows theeffect of antibody treatment on total plasmablast(CD3-CD20-CD27hiCD38hi) number. Each line represents an individualpatient (fourteen patients represented).

FIG. 23. Baseline circulating plasmablasts. CD19+ plasmablasts per 10⁴CD19+ B cells are shown for eleven healthy donors and fourteen IgG4-RDpatients prior to treatment. Plasmablasts are elevated in active IgG4-RDpatients as compared to healthy donors.

FIGS. 24A-24G. Flow cytometry gating strategy for CD4 CTLs.

FIGS. 25A-25B. FIG. 25A shows the effect of anti-CD19 antibodyS267E/L328F on SLAMF7+CD4 CTL (CD4+CD45RO+CD27-SLAMF7+) number. FIG. 25Bshows the effect of anti-CD19 antibody S267E/L328F on effector CD4 CTL(CD4+CD45RO+CD27-SLAMF7+CD57+CD28−) number. Each line represents anindividual patient.

FIGS. 26A-26D. Flow cytometry gating strategy for apoptosis assay.

FIGS. 27A-27L. Apoptosis Assay. No significant apoptosis of B cells(FIG. 27A-27D), CD4+ T cells (FIG. 27E-27H), or CD8+ T cells (FIG.271-27L) was observed in patients after receiving anti-CD19 antibodyS267E/L328F therapy at days 1, 8, 15, and 29.

FIGS. 28A-28L. Anti-CD19 antibody S267E/L328F blocks BCR linkedsignaling pathways. Phosphorylation of BTK, AKT, ERK, and SYK wereexamined before treatment with anti-CD19 antibody S267E/L328F (FIG.28A-28D), 2 hours after treatment (FIG. 28E-28H), and 24 hours aftertreatment (FIG. 28I-28L). Yellow represents the FMO control; blue, notreatment; and red F(ab)2 (IgM+IgG) treated.

FIG. 29. CD4+SLAMF7+ CTL numbers were increased in the peripheral bloodof IgG4-RD patients as compared to controls.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The humoral immune response (e.g., the result of diverse B cellresponses) may be initiated when B cells are activated by an antigen andsubsequently differentiated into plasma cells. Binding of membrane boundB cell receptor (BCR) on B cells by an antigen activates anintracellular signaling cascade, including calcium mobilization, whichleads to cell proliferation and differentiation. Coengagement of cognateBCR) with the inhibitory Fc receptor (FcγRIIb) inhibits B cellactivation signals through a negative feedback loop.

The importance of FcγRIIb in negative regulation of B cell responses hasbeen demonstrated using FcγRIIb-deficient mice, which fail to regulatehumoral responses (Wernersson, S. et al., 1999, J. Immunol. 163,618-622), are sensitized to collagen-induced arthritis (Yuasa, T. etal., 1999, J. Exp. Med. 189, 187-194), and develop lupus-like disease(Fukuyama, H. et al., J. V., 2005, Nat. Immunol. 6, 99-106; McGaha, T.L. et al., 2005, Science 307, 590-593) and Goodpasture's syndrome(Nakamura, A. et al., 2000, J. Exp. Med. 191, 899-906). FcγRIIbdysregulation has also been associated with human autoimmune disease.For example, polymorphisms in the promoter (Blank, M. C. et al., 2005,Hum. Genet. 117, 220-227; Olferiev, M. et al., 2007, J. Biol. Chem. 282,1738-1746) and transmembrane domain (Chen, J. Y. et al., 2006, ArthritisRheum. 54, 3908-3917; Floto, R. A. et al., Nat. Med. 11, 1056-1058; Li,X. et al., 2003, Arthritis Rheum. 48, 3242-3252) of FcγRIIb have beenlinked with increased prevalence of systemic lupus erythematosus (SLE).SLE patients also show reduced FcγRIIb surface expression on B cells(Mackay, M. et al., 2006, J. Exp. Med. 203, 2157-2164; Su, K. et al.,2007, J. Immunol. 178, 3272-3280) and, as a consequence, exhibitdysregulated calcium signaling (Mackay, M. et al., 2006, J. Exp. Med.203, 2157-2164). The pivotal role of FcγRIIb in regulating B cells,supported by mouse models and clinical evidence, makes it an attractivetherapeutic target for controlling autoimmune and inflammatory disorders(Pritchard, N. R. & Smith, K. G., 2003, Immunology 108, 263-273;Ravetch, J. V. & Lanier, L. L., 2000, Science 290, 84-89; Stefanescu, R.N. et al., 2004, J. Clin. Immunol. 24, 315-326).

Described herein are antibodies that mimic the inhibitory effects ofcoengagement of cognate BCR with FcγRIIb on B cells. For example,describe herein are variant anti-CD19 antibodies engineered such thatthe Fc domain binds to FcγRIIb with up to ˜430-fold greater affinity.Relative to native IgG1, the FcγRIIb binding-enhanced (IIbE) variantsstrongly inhibit BCR-induced calcium mobilization and viability inprimary human B cells. Inhibitory effects involved phosphorylation ofSH2-containing inositol polyphosphate 5-phosphatase (SHIP), which isknown to be involved in FcγRIIb-induced negative feedback of B cellactivation. Coengagement of BCR and FcγRIIb by IIbE variants alsoovercame the anti-apoptotic effects of BCR activation. The use of asingle antibody to suppress B cell functions by coengagement of cognateBCR and FcγRIIb may represent a novel approach in the treatment of Bcell-mediated diseases. A nonlimiting example of B cell-mediateddiseases includes IgG4-related disease.

Described herein are several definitions. Such definitions are meant toencompass grammatical equivalents.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “antibody” herein is meant a protein consisting of one or morepolypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (κ), lambda (λ), and heavy chain geneticloci, which together comprise the myriad variable region genes, and theconstant region genes mu (υ), delta (δ), gamma (γ), sigma (σ), and alpha(α) which encode the IgM, IgD, IgG (IgG1, IgG2, IgG3, and IgG4), IgE,and IgA (IgA1 and IgA2) isotypes respectively. Antibody herein is meantto include full length antibodies and antibody fragments, and may referto a natural antibody from any organism, an engineered antibody, or anantibody generated recombinantly for experimental, therapeutic, or otherpurposes.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids or any non-natural analogues thatmay be present at a specific, defined position.

By “CD32b⁺ cell” or “FcγRIIb⁺ cell” as used herein is meant any cell orcell type that expresses CD32b (FcγRIIb). CD32b+ cells include but arenot limited to B cells, plasma cells, dendritic cells, macrophages,neutrophils, mast cells, basophils, or eosinophils.

By “CDC” or “complement dependent cytotoxicity” as used herein is meantthe reaction wherein one or more complement protein components recognizebound antibody on a target cell and subsequently cause lysis of thetarget cell.

By “constant region” of an antibody as defined herein is meant theregion of the antibody that is encoded by one of the light or heavychain immunoglobulin constant region genes. By “constant light chain” or“light chain constant region” as used herein is meant the region of anantibody encoded by the kappa (Cκ) or lambda (Cλ) light chains. Theconstant light chain typically comprises a single domain, and as definedherein refers to positions 108-214 of OK or Cλ, wherein numbering isaccording to the EU index. By “constant heavy chain” or “heavy chainconstant region” as used herein is meant the region of an antibodyencoded by the mu, delta, gamma, alpha, or epsilon genes to define theantibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For fulllength IgG antibodies, the constant heavy chain, as defined herein,refers to the N-terminus of the CH1 domain to the C-terminus of the CH3domain, thus comprising positions 118-447, wherein numbering isaccording to the EU index.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include FcγR-mediated effectorfunctions such as ADCC and ADCP, and complement-mediated effectorfunctions such as CDC. Further, effector functions includeFcγRIIb-mediated effector functions, such as inhibitory functions (e.g.,downregulating, reducing, inhibiting etc., B cell responses, e.g., ahumoral immune response).

By “effector cell” as used herein is meant a cell of the immune systemthat expresses one or more Fc and/or complement receptors and mediatesone or more effector functions. Effector cells include but are notlimited to monocytes, macrophages, neutrophils, dendritic cells,eosinophils, mast cells, platelets, B cells, large granular lymphocytes,Langerhans' cells, natural killer (NK) cells, and γδ T cells, and may befrom any organism including but not limited to humans, mice, rats,rabbits, and monkeys.

By “Fab” or “Fab region” as used herein is meant the polypeptides thatcomprise the V_(H), CH1, V_(H), and C_(L) immunoglobulin domains. Fabmay refer to this region in isolation, or this region in the context ofa full length antibody or antibody fragment.

By “Fc” or “Fc region,” as used herein is meant the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain and in some cases, part of thehinge. Thus Fc refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM, and the flexible hinge N-terminalto these domains. For IgA and IgM, Fc may include the J chain. For IgG,Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3)and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. Fc may refer to this region in isolation, or this region inthe context of an Fc polypeptide, as described below.

By “Fc polypeptide” as used herein is meant a polypeptide that comprisesall or part of an Fc region. Fc polypeptides include antibodies, Fcfusions, isolated Fcs, and Fc fragments. Immunoglobulins may be Fcpolypeptides.

By “Fc fusion” as used herein is meant a protein wherein one or morepolypeptides is operably linked to Fc. Fc fusion is herein meant to besynonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”,and “receptor globulin” (sometimes with dashes) as used in the prior art(Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al.,1997, Curr Opin Immunol 9:195-200, both hereby entirely incorporated byreference). An Fc fusion combines the Fc region of an immunoglobulinwith a fusion partner, which in general may be any protein, polypeptide,or small molecule. The role of the non-Fc part of an Fc fusion, i.e.,the fusion partner, is to mediate target binding, and thus it isfunctionally analogous to the variable regions of an antibody. Virtuallyany protein or small molecule may be linked to Fc to generate an Fcfusion. Protein fusion partners may include, but are not limited to, thetarget-binding region of a receptor, an adhesion molecule, a ligand, anenzyme, a cytokine, a chemokine, or some other protein or proteindomain. Small molecule fusion partners may include any therapeutic agentthat directs the Fc fusion to a therapeutic target. Such targets may beany molecule, e.g., an extracellular receptor that is implicated indisease.

By “Fc gamma receptor” or “FcγR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and aresubstantially encoded by the FcγR genes. In humans this family includesbut is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb,and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65,incorporated entirely by reference), as well as any undiscovered humanFcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism,including but not limited to humans, mice, rats, rabbits, and monkeys.Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32),FcγRII (CD16), and FcγRII-2 (CD16-2), as well as any undiscovered mouseFcγRs or FcγR isoforms or allotypes.

By “Fc ligand” or “Fc receptor” as used herein is meant a molecule,e.g., a polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc-ligand complex. Fc ligands include but are notlimited to FcγRs, FcγRs, FcγRs, FcRn, C1q, C3, mannan binding lectin,mannose receptor, staphylococcal protein A, streptococcal protein G, andviral FcγR. Fc ligands also include Fc receptor homologs (FcRH), whichare a family of Fc receptors that are homologous to the FcγRs (Davis etal., 2002, Immunological Reviews 190:123-136). Fc ligands may includeundiscovered molecules that bind Fc.

By “full length antibody” as used herein is meant the structure thatconstitutes the natural biological form of an antibody, includingvariable and constant regions. For example, in most mammals, includinghumans and mice, the full length antibody of the IgG isotype is atetramer and consists of two identical pairs of two immunoglobulinchains, each pair having one light and one heavy chain, each light chaincomprising immunoglobulin domains VL and CL, and each heavy chaincomprising immunoglobulin domains VH, Cγ1, Cγ2, and Cγ3. In somemammals, for example in camels and llamas, IgG antibodies may consist ofonly two heavy chains, each heavy chain comprising a variable domainattached to the Fc region.

By “immunoglobulin” herein is meant a protein comprising one or morepolypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include but are not limited to antibodies (includingbispecific antibodies) and Fc fusions. Immunoglobulins may have a numberof structural forms, including but not limited to full lengthantibodies, antibody fragments, and individual immunoglobulin domains.

By “immunoglobulin (Ig) domain” as used herein is meant a region of animmunoglobulin that exists as a distinct structural entity asascertained by one skilled in the art of protein structure. Ig domainstypically have a characteristic f3-sandwich folding topology. The knownIg domains in the IgG isotype of antibodies are VH Cyt, Cγ2, Cγ3, VL,and CL.

By “IgG” or “IgG immunoglobulin” as used herein is meant a polypeptidebelonging to the class of antibodies that are substantially encoded by arecognized immunoglobulin gamma gene. In humans this class comprises thesubclasses or isotypes IgG1, IgG2, IgG3, and IgG4. By “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Theknown human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1,IgA2, IgM, IgD, and IgE.

By “modification” herein is meant an alteration in the physical,chemical, or sequence properties of a protein, polypeptide, antibody, orimmunoglobulin. Modifications described herein include amino acidmodifications and glycoform modifications.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. By “amino acidsubstitution” or “substitution” herein is meant the replacement of anamino acid at a particular position in a parent polypeptide sequencewith another amino acid. For example, the substitution S267E refers to avariant polypeptide, in this case a constant heavy chain variant, inwhich the serine at position 267 is replaced with glutamic acid. By“amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid at a particular position in a parentpolypeptide sequence. By “amino acid deletion” or “deletion” as usedherein is meant the removal of an amino acid at a particular position ina parent polypeptide sequence.

By “glycoform modification” or “modified glycoform” or “engineeredglycoform” as used herein is meant a carbohydrate composition that iscovalently attached to a protein, for example an antibody, wherein saidcarbohydrate composition differs chemically from that of a parentprotein. Modified glycoform typically refers to the differentcarbohydrate or oligosaccharide; thus for example an Fc variant maycomprise a modified glycoform. Alternatively, modified glycoform mayrefer to the Fc variant that comprises the different carbohydrate oroligosaccharide.

By “parent polypeptide,” “parent protein,” “parent immunoglobulin,”“precursor polypeptide,” “precursor protein,” or “precursorimmunoglobulin” as used herein is meant an unmodified polypeptide,protein, or immunoglobulin that is subsequently modified to generate avariant, e.g., any polypeptide, protein, or immunoglobulin which servesas a template and/or basis for at least one amino acid modificationdescribed herein. The parent polypeptide may be a naturally occurringpolypeptide, or a variant or engineered version of a naturally occurringpolypeptide. Parent polypeptide may refer to the polypeptide itself,compositions that comprise the parent polypeptide, or the amino acidsequence that encodes it. Accordingly, by “parent Fc polypeptide” asused herein is meant an Fc polypeptide that is modified to generate avariant Fc polypeptide, and by “parent antibody” as used herein is meantan antibody that is modified to generate a variant antibody (e.g., aparent antibody may include, but is not limited to, a protein comprisingthe constant region of a naturally occurring Ig).

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index as in Kabat. For example,position 297 is a position in the human antibody IgG1.

By “polypeptide” or “protein” as used herein is meant at least twocovalently attached amino acids, which includes proteins, polypeptides,oligopeptides, and peptides.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297, also referred to as N297) is a residue in thehuman antibody IgG1.

By “target antigen” as used herein is meant the molecule that is boundby the variable region of a given antibody, or the fusion partner of anFc fusion. A target antigen may be a protein, carbohydrate, lipid, orother chemical compound. An antibody or Fc fusion is said to be“specific” for a given target antigen based on having affinity for thetarget antigen.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the VK, VX, and/or VH genes that make up the kappa,lambda, and heavy chain immunoglobulin genetic loci respectively.

By “variant polypeptide,” “polypeptide variant,” or “variant” as usedherein is meant a polypeptide sequence that differs from that of aparent polypeptide sequence by virtue of at least one amino acidmodification. The parent polypeptide may be a naturally occurring orwild-type (WT) polypeptide, or may be a modified version of a WTpolypeptide. Variant polypeptide may refer to the polypeptide itself, acomposition comprising the polypeptide, or the amino sequence thatencodes it. In some embodiments, variant polypeptides disclosed herein(e.g., variant immunoglobulins) may have at least one amino acidmodification compared to the parent polypeptide, e.g. from about one toabout ten amino acid modifications, from about one to about five aminoacid modifications, etc. compared to the parent. The variant polypeptidesequence herein may possess at least about 80% homology with a parentpolypeptide sequence, e.g., at least about 90% homology, 95% homology,etc. Accordingly, by “Fc variant” or “variant Fc” as used herein ismeant an Fc sequence that differs from that of a parent Fc sequence byvirtue of at least one amino acid modification. An Fc variant may onlyencompass an Fc region, or may exist in the context of an antibody, Fcfusion, isolated Fc, Fc fragment, or other polypeptide that issubstantially encoded by Fc. Fc variant may refer to the Fc polypeptideitself, compositions comprising the Fc variant polypeptide, or the aminoacid sequence that encodes it. By “Fc polypeptide variant” or “variantFc polypeptide” as used herein is meant an Fc polypeptide that differsfrom a parent Fc polypeptide by virtue of at least one amino acidmodification. By “protein variant” or “variant protein” as used hereinis meant a protein that differs from a parent protein by virtue of atleast one amino acid modification. By “antibody variant” or “variantantibody” as used herein is meant an antibody that differs from a parentantibody by virtue of at least one amino acid modification. By “IgGvariant” or “variant IgG” as used herein is meant an antibody thatdiffers from a parent IgG by virtue of at least one amino acidmodification. By “immunoglobulin variant” or “variant immunoglobulin” asused herein is meant an immunoglobulin sequence that differs from thatof a parent immunoglobulin sequence by virtue of at least one amino acidmodification.

By “wild type” or “WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein, polypeptide, antibody, immunoglobulin, IgG,etc. has an amino acid sequence or a nucleotide sequence that has notbeen intentionally modified.

Immunoglobulins

As described herein, an immunoglobulin may be an antibody, an Fc fusion,an isolated Fc, an Fc fragment, or an Fc polypeptide. In one embodiment,an immunoglobulin is an antibody.

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. The light and heavy chainvariable regions show significant sequence diversity between antibodies,and are responsible for binding the target antigen. Each chain is madeup of individual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. Heavy chains are classifiedas mu, delta, gamma, alpha, or epsilon, and define the antibody'sisotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has severalsubclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4.IgM has subclasses, including, but not limited to, IgM1 and IgM2. IgAhas several subclasses, including but not limited to IgA1 and IgA2.Thus, “isotype” as used herein is meant any of the classes andsubclasses of immunoglobulins defined by the chemical and antigeniccharacteristics of their constant regions. The known humanimmunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1,IgM2, IgD, and IgE.

Each of the light and heavy chains is made up of two distinct regions,referred to as the variable and constant regions. The IgG heavy chain iscomposed of four immunoglobulin domains linked from N- to C-terminus inthe order VH-CH1-CH2-CH3, referring to the heavy chain variable domain,heavy chain constant domain 1, heavy chain constant domain 2, and heavychain constant domain 3 respectively (also referred to asVH-Cγ1-Cγ2-Cγ3, referring to the heavy chain variable domain, constantgamma 1 domain, constant gamma 2 domain, and constant gamma 3 domainrespectively). The IgG light chain is composed of two immunoglobulindomains linked from N- to C-terminus in the order VL-CL, referring tothe light chain variable domain and the light chain constant domainrespectively. The constant regions show less sequence diversity, and areresponsible for binding a number of natural proteins to elicit importantbiochemical events. The distinguishing features between these antibodyclasses are their constant regions, although subtler differences mayexist in the variable region.

The variable region of an antibody contains the antigen bindingdeterminants of the molecule, and thus determines the specificity of anantibody for its target antigen. The variable region is so named becauseit is the most distinct in sequence from other antibodies within thesame class. The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.There are 6 CDRs total, three each per heavy and light chain, designatedVH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variableregion outside of the CDRs is referred to as the framework (FR) region.Although not as diverse as the CDRs, sequence variability does occur inthe FR region between different antibodies. Overall, this characteristicarchitecture of antibodies provides a stable scaffold (the FR region)upon which substantial antigen binding diversity (the CDRs) can beexplored by the immune system to obtain specificity for a broad array ofantigens. A number of high-resolution structures are available for avariety of variable region fragments from different organisms, someunbound and some in complex with antigen. Sequence and structuralfeatures of antibody variable regions are disclosed, for example, inMorea et al., 1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods20:267-279, hereby entirely incorporated by reference, and the conservedfeatures of antibodies are disclosed, for example, in Maynard et al.,2000, Annu Rev Biomed Eng 2:339-376, hereby entirely incorporated byreference.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. In the IgG subclass ofimmunoglobulins, there are several immunoglobulin domains in the heavychain. By “immunoglobulin (Ig) domain” herein is meant a region of animmunoglobulin having a distinct tertiary structure. Of interest inembodiments described herein are the heavy chain domains, including, theconstant heavy (CH) domains and the hinge region. In the context of IgGantibodies, the IgG isotypes each have three CH regions. Accordingly,“CH” domains in the context of IgG are as follows: “CH1” refers topositions 118-220 according to the EU index as in Kabat. “CH2” refers topositions 237-340 according to the EU index as in Kabat, and “CH3”refers to positions 341-447 according to the EU index as in Kabat.

Another important region of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230 to 236.

Of interest in embodiments described herein are the Fc regions. By “Fc”or “Fc region,” as used herein is meant the polypeptide comprising theconstant region of an antibody excluding the first constant regionimmunoglobulin domain and in some cases, part of the hinge. Thus Fcrefers to the last two constant region immunoglobulin domains of IgA,IgD, and IgG, and the last three constant region immunoglobulin domainsof IgE and IgM, and the flexible hinge N-terminal to these domains. ForIgA and IgM, Fc may include the J chain. For IgG, Fc comprisesimmunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the lowerhinge region between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to include residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. Fc may refer to this region in isolation, or this region inthe context of an Fc polypeptide, as described below. By “Fcpolypeptide” as used herein is meant a polypeptide that comprises all orpart of an Fc region. Fc polypeptides include antibodies, Fc fusions,isolated Fcs, and Fc fragments.

The Fc region of an antibody interacts with a number of Fc receptors andligands, imparting an array of important functional capabilitiesreferred to as effector functions. For IgG the Fc region, Fc comprisesIg domains Cγ2 and Cγ3 and the N-terminal hinge leading into Cγ2. Animportant family of Fc receptors for the IgG class are the Fc gammareceptors (FcγRs). These receptors mediate communication betweenantibodies and the cellular arm of the immune system (Raghavan et al.,1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu RevImmunol 19:275-290, both hereby entirely incorporated by reference). Inhumans this protein family includes FcγRI (CD64), including isoformsFcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa(including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 andFcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa(including allotypes V158 and F158) and FcγRIIIb (including allotypesFcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett82:57-65, hereby entirely incorporated by reference). These receptorstypically have an extracellular domain that mediates binding to Fc, amembrane spanning region, and an intracellular domain that may mediatesome signaling event within the cell. These receptors are expressed in avariety of immune cells including monocytes, macrophages, neutrophils,dendritic cells, eosinophils, mast cells, platelets, B cells, largegranular lymphocytes, Langerhans' cells, natural killer (NK) cells, andyy T cells. Formation of the Fc/FcγR complex recruits these effectorcells to sites of bound antigen, typically resulting in signaling eventswithin the cells and important subsequent immune responses such asrelease of inflammation mediators, B cell activation, endocytosis,phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic andphagocytic effector functions is a potential mechanism by whichantibodies destroy targeted cells. The cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell isreferred to as antibody dependent cell-mediated cytotoxicity (ADCC)(Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie etal., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu RevImmunol 19:275-290, both hereby entirely incorporated by reference). Thecell-mediated reaction wherein nonspecific cytotoxic cells that expressFcγRs recognize bound antibody on a target cell and subsequently causephagocytosis of the target cell is referred to as antibody dependentcell-mediated phagocytosis (ADCP).

The different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett82:57-65, hereby entirely incorporated by reference). The FcγRs bind theIgG Fc region with different affinities. The extracellular domains ofFcγRIIIa and FcγRIIIb are 96% identical, however FcγRIIIb does not havea intracellular signaling domain. Furthermore, whereas FcγRI, FcγRIIa/c,and FcγRIIIa are positive regulators of immune complex-triggeredactivation, characterized by having an intracellular domain that has animmunoreceptor tyrosine-based activation motif (ITAM), FcγRIIb has animmunoreceptor tyrosine-based inhibition motif (ITIM) and is thereforeinhibitory. Thus the former are referred to as activation receptors, andFcγRIIb is referred to as an inhibitory receptor. Despite thesedifferences in affinities and activities, all FcγRs bind the same regionon Fc, at the N-terminal end of the Cγ2 domain and the preceding hinge.This interaction is well characterized structurally (Sondermann et al.,2001, J Mol Biol 309:737-749, hereby entirely incorporated byreference), and several structures of the human Fc bound to theextracellular domain of human FcγRIIIb have been solved (pdb accessioncode 1E4K) (Sondermann et al., 2000, Nature 406:267-273, hereby entirelyincorporated by reference) (pdb accession codes 1IIS and 1IIX) (Radaevet al., 2001, J Biol Chem 276:16469-16477, hereby entirely incorporatedby reference).

An overlapping but separate site on Fc serves as the interface for thecomplement protein C1q. In the same way that Fc/FcγR binding mediatesADCC, Fc/C1q binding mediates complement dependent cytotoxicity (CDC). Asite on Fc between the Cγ2 and Cγ3 domains mediates interaction with theneonatal receptor FcRn, the binding of which recycles endocytosedantibody from the endosome back to the bloodstream (Raghavan et al.,1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu RevImmunol 18:739-766, both hereby entirely incorporated by reference).This process, coupled with preclusion of kidney filtration due to thelarge size of the full length molecule, results in favorable antibodyserum half-lives ranging from one to three weeks. Binding of Fc to FcRnalso plays a key role in antibody transport. The binding site for FcRnon Fc is also the site at which the bacterial proteins A and G bind. Thetight binding by these proteins is typically exploited as a means topurify antibodies by employing protein A or protein G affinitychromatography during protein purification. The fidelity of theseregions, the complement and FcRn/protein A binding regions are importantfor both the clinical properties of antibodies and their development.

A key feature of the Fc region is the conserved N-linked glycosylationthat occurs at N297. This carbohydrate, or oligosaccharide as it issometimes referred, plays a critical structural and functional role forthe antibody, and is one of the principle reasons that antibodies mustbe produced using mammalian expression systems. Efficient Fc binding toFcγR and C1q requires this modification, and alterations in thecomposition of the N297 carbohydrate or its elimination affect bindingto these proteins (Umana et al., 1999, Nat Biotechnol 17:176-180; Davieset al., 2001, Biotechnol Bioeng 74:288-294; Mimura et al., 2001, J BiolChem 276:45539-45547; Radaev et al., 2001, J Biol Chem 276:16478-16483;Shields et al., 2001, J Biol Chem 276:6591-6604; Shields et al., 2002, JBiol Chem 277:26733-26740; Simmons et al., 2002, J Immunol Methods263:133-147, all hereby entirely incorporated by reference).

Immunoglobulins of embodiments described herein may also be anantibody-like protein referred to as an Fc fusion (Chamow et al., 1996,Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol9:195-200, both incorporated entirely by reference). “Fc fusion” isherein meant to be synonymous with the terms “immunoadhesin”, “Igfusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes)as used in the prior art (Chamow et al., 1996, Trends Biotechnol14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200). An Fcfusion is a protein wherein one or more polypeptides, herein referred toas a “fusion partner,” is operably linked to Fc. An Fc fusion combinesthe Fc region of an antibody, and thus its favorable effector functionsand pharmacokinetics, with the target-binding region of a receptor,ligand, or some other protein or protein domain. The role of the latteris to mediate target recognition, and thus it is functionally analogousto the antibody variable region. Because of the structural andfunctional overlap of Fc fusions with antibodies, the discussion onantibodies in the present disclosure extends also to Fc fusions.

Virtually any protein or small molecule may be linked to Fc to generatean Fc fusion. Protein fusion partners may include, but are not limitedto, the variable region of any antibody, the target-binding region of areceptor, an adhesion molecule, a ligand, an enzyme, a cytokine, achemokine, or some other protein or protein domain. Small moleculefusion partners may include any agent that directs the Fc fusion to atarget antigen. Such target antigen may be any molecule, e.g., anextracellular receptor, that is implicated in disease. Fc fusions ofembodiments described herein may target virtually antigen that isexpressed on CD32b⁺ cells.

Fusion partners may be linked to any region of an Fc region, includingat the N- or C-termini, or at some residue in-between the termini. Inone embodiment, a fusion partner is linked at the N- or C-terminus ofthe Fc region. A variety of linkers may find use in some embodimentsdescribed herein to covalently link Fc regions to a fusion partner. By“linker”, “linker sequence”, “spacer”, “tethering sequence” orgrammatical equivalents thereof, herein is meant a molecule or group ofmolecules (such as a monomer or polymer) that connects two molecules andoften serves to place the two molecules in a configuration. Linkers areknown in the art; for example, homo- or hetero-bifunctional linkers asare well known (see, 1994 Pierce Chemical Company catalog, technicalsection on cross-linkers, pages 155-200, incorporated entirely byreference). A number of strategies may be used to covalently linkmolecules together. These include, but are not limited to polypeptidelinkages between N- and C-termini of proteins or protein domains,linkage via disulfide bonds, and linkage via chemical cross-linkingreagents. In one aspect of this embodiment, the linker is a peptidebond, generated by recombinant techniques or peptide synthesis. Thelinker peptide may predominantly include the following amino acidresidues: Gly, Ser, Ala, or Thr. The linker peptide should have a lengththat is adequate to link two molecules in such a way that they assumethe correct conformation relative to one another so that they retain thedesired activity. Suitable lengths for this purpose include at least oneand not more than 50 amino acid residues. In one embodiment, the linkeris from about 1 to 30 amino acids in length. In one embodiment, hlinkers of 1 to 20 amino acids in length may be used. Useful linkersinclude glycine-serine polymers (including, for example, (GS)n, (GSGGS)n(set forth as SEQ ID NO:1), (GGGGS)n (set forth as SEQ ID NO:2), and(GGGS)n (set forth as SEQ ID NO:3), where n is an integer of at leastone), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers, as will be appreciated by those in the art.Alternatively, a variety of nonproteinaceous polymers, including but notlimited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may find use as linkers, that is may find use to link an Fcregions to a fusion partner.

Also contemplated as fusion partners are Fc polypeptides. Thus animmunoglobulin as described herein may be a multimeric Fc polypeptide,comprising two or more Fc regions. The advantage of such a molecule isthat it provides multiple binding sites for Fc receptors with a singleprotein molecule. In one embodiment, Fc regions may be linked using achemical engineering approach. For example, Fab's and Fc's may be linkedby thioether bonds originating at cysteine residues in the hinges,generating molecules such as FabFc₂. Fc regions may be linked usingdisulfide engineering and/or chemical cross-linking. In one embodiment,Fc regions may be linked genetically. In one embodiment, Fc regions inan immunoglobulin are linked genetically to generated tandemly linked Fcregions as described in U.S. Patent Publication No. 2005/0249723,incorporated entirely by reference. Tandemly linked Fc polypeptides maycomprise two or more Fc regions, e.g., one to three Fc regions, two Fcregions. It may be advantageous to explore a number of engineeringconstructs in order to obtain homo- or hetero-tandemly linked Fc regionswith the most favorable structural and functional properties. Tandemlylinked Fc regions may be homo-tandemly linked Fc regions, that is an Fcregion of one isotype is fused genetically to another Fc region of thesame isotype. It is anticipated that because there are multiple FcγR,C1q, and/or FcRn binding sites on tandemly linked Fc polypeptides,effector functions and/or pharmacokinetics may be enhanced. In analternate embodiment, Fc regions from different isotypes may be tandemlylinked, referred to as hetero-tandemly linked Fc regions. For example,because of the capacity to target FcγR and FcαRI receptors, animmunoglobulin that binds both FcγRs and FcαRI may provide a significantclinical improvement.

The immunoglobulins of embodiments disclosed herein may be substantiallyencoded by immunoglobulin genes belonging to any of the antibodyclasses. In certain embodiments, the immunoglobulins disclosed hereinfind use in antibodies or Fc fusions that comprise sequences belongingto the IgG class of antibodies, including IgG1, IgG2, IgG3, or IgG4.FIG. 1 provides an alignment of these human IgG sequences. In alternateembodiments, immunoglobulins disclosed herein find use in antibodies orFc fusions that comprise sequences belonging to the IgA (includingsubclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classes of antibodies.The immunoglobulins disclosed herein may comprise more than one proteinchain, e.g., may be an antibody or Fc fusion that is a monomer or anoligomer, including a homo- or hetero-oligomer.

Immunoglobulins disclosed herein may be substantially encoded by genesfrom any organism, e.g., mammals (including, but not limited to humans,rodents (including but not limited to mice and rats), lagomorpha(including but not limited to rabbits and hares), camelidae (includingbut not limited to camels, llamas, and dromedaries), and non-humanprimates, including but not limited to Prosimians, Platyrrhini (NewWorld monkeys), Cercopithecoidea (Old World monkeys), and Hominoideaincluding the Gibbons and Lesser and Great Apes. In a certainembodiments, the immunoglobulins disclosed herein may be substantiallyhuman.

As is well known in the art, immunoglobulin polymorphisms exist in thehuman population. Gm polymorphism is determined by the IGHG1, IGHG2 andIGHG3 genes which have alleles encoding allotypic antigenic determinantsreferred to as G1m, G2m, and G3m allotypes for markers of the humanIgG1, IgG2 and IgG3 molecules (no Gm allotypes have been found on thegamma 4 chain). Markers may be classified into ‘allotypes’ and‘isoallotypes.’ These are distinguished on different serological basesdependent upon the strong sequence homologies between isotypes.Allotypes are antigenic determinants specified by allelic forms of theIg genes. Allotypes represent slight differences in the amino acidsequences of heavy or light chains of different individuals. Even asingle amino acid difference can give rise to an allotypic determinant,although in many cases there are several amino acid substitutions thathave occurred. Allotypes are sequence differences between alleles of asubclass whereby the antisera recognize only the allelic differences. Anisoallotype is an allele in one isotype which produces an epitope whichis shared with a non-polymorphic homologous region of one or more otherisotypes and because of this the antisera will react with both therelevant allotypes and the relevant homologous isotypes (Clark, 1997,IgG effector mechanisms, Chem Immunol. 65:88-110; Gorman & Clark, 1990,Semin Immunol 2(6):457-66, both hereby entirely incorporated byreference).

Allelic forms of human immunoglobulins have been well-characterized (WHOReview of the notation for the allotypic and related markers of humanimmunoglobulins. J Immunogen 1976, 3: 357-362; WHO Review of thenotation for the allotypic and related markers of human immunoglobulins.1976, Eur. J. Immunol. 6, 599-601; Loghem E van, 1986, Allotypicmarkers, Monogr Allergy 19: 40-51, all hereby entirely incorporated byreference). Additionally, other polymorphisms have been characterized(Kim et al., 2001, J. Mol. Evol. 54:1-9, hereby entirely incorporated byreference). At present, 18 Gm allotypes are known: G1m (1, 2, 3, 17) orG1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15,16, 21, 24, 26, 27, 28) or G3m (b1, c3, b5, b0, b3, b4, s, t, g1, c5, u,v, g5) (Lefranc, et al., The human IgG subclasses: molecular analysis ofstructure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990);Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211, both hereby entirelyincorporated by reference). Allotypes that are inherited in fixedcombinations are called Gm haplotypes. FIG. 2 shows common haplotypes ofthe gamma chain of human IgG1 (FIG. 2A) and IgG2 (FIG. 2A) showing thepositions and the relevant amino acid substitutions. The immunoglobulinsdisclosed herein may be substantially encoded by any allotype,isoallotype, or haplotype of any immunoglobulin gene.

The immunoglobulins disclosed herein may compose an Fc polypeptide,including but not limited to antibodies, isolated Fcs, Fc fragments, andFc fusions. In one embodiment, an immunoglobulin disclosed herein is afull length antibody, constituting the natural biological form of anantibody, including variable and constant regions. For the IgG isotypefull length antibody is a tetramer and consists of two identical pairsof two immunoglobulin chains, each pair having one light and one heavychain, each light chain comprising immunoglobulin domains VL and CL, andeach heavy chain comprising immunoglobulin domains VH, Cyt, Cγ2, andCγ3. In another embodiment, immunoglobulins disclosed herein areisolated Fc regions or Fc fragments.

Immunoglobulins disclosed herein may be a variety of structures,including, but not limited antibody fragments, bispecific antibodies,minibodies, domain antibodies, synthetic antibodies (sometimes referredto herein as “antibody mimetics”), chimeric antibodies, humanizedantibodies, antibody fusions (sometimes referred to as “antibodyconjugates”), and fragments of each, respectively.

In one embodiment, the antibody is an antibody fragment. Specificantibody fragments include, but are not limited to, (i) the Fab fragmentconsisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment,which consists of a single variable, (v) isolated CDR regions, (vi)F(ab′)2 fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site, (viii) bispecificsingle chain Fv dimers, and (ix) “diabodies” or “triabodies”,multivalent or multispecific fragments constructed by gene fusion. Theantibody fragments may be modified. For example, the molecules may bestabilized by the incorporation of disulphide bridges linking the VH andVL domains. Examples of antibody formats and architectures are describedin Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136, andCarter 2006, Nature Reviews Immunology 6:343-357 and references citedtherein, all expressly incorporated by reference.

In one embodiment, an antibody disclosed herein may be a multispecificantibody, and notably a bispecific antibody, also sometimes referred toas “diabodies.” These are antibodies that bind to two (or more)different antigens. Diabodies can be manufactured in a variety of waysknown in the art, e.g., prepared chemically or from hybrid hybridomas.In one embodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. In somecases, the scFv can be joined to the Fc region, and may include some orall of the hinge region. For a description of multispecific antibodiessee Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136 andreferences cited therein, all expressly incorporated by reference.

Nonhuman, Chimeric, Humanized, and Fully Human Antibodies

The variable region of an antibody, as is well known in the art, cancompose sequences from a variety of species. In some embodiments, theantibody variable region can be from a nonhuman source, including butnot limited to mice, rats, rabbits, camels, llamas, and monkeys. In someembodiments, the scaffold components can be a mixture from differentspecies. As such, an antibody disclosed herein may be a chimericantibody and/or a humanized antibody. In general, both “chimericantibodies” and “humanized antibodies” refer to antibodies that combineregions from more than one species. For example, “chimeric antibodies”traditionally comprise variable region(s) from a mouse or other nonhumanspecies and the constant region(s) from a human.

“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. No. 5,693,762, incorporated entirely by reference.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein). Humanization or other methods of reducing theimmunogenicity of nonhuman antibody variable regions may includeresurfacing methods, as described for example in Roguska et al., 1994,Proc. Natl. Acad. Sci. USA 91:969-973. In one embodiment, the parentantibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, that is, to increase the affinity ofthe variable region for its target antigen. Other humanization methodsmay involve the grafting of only parts of the CDRs, including but notlimited to methods described in U.S. Ser. No. 09/810,502; Tan et al.,2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol.169:3076-3084. Structure-based methods may be employed for humanizationand affinity maturation, for example as described in U.S. Pat. No.7,117,096, and related applications, all incorporated entirely byreference. In certain variations, the immunogenicity of the antibody isreduced using a method described in U.S. Pat. No. 7,657,380,incorporated entirely by reference.

In one embodiment, the antibody is a fully human antibody with at leastone modification as outlined herein. “Fully human antibody” or “completehuman antibody” refers to a human antibody having the gene sequence ofan antibody derived from a human chromosome with the modificationsoutlined herein. Fully human antibodies may be obtained, for example,using transgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol8:455-458) or human antibody libraries coupled with selection methods(Griffiths et al., 1998, Curr Opin Biotechnol 9:102-108).

Target Antigen

In one embodiment, an antigen may be targeted by the immunoglobulinsdisclosed herein, including but not limited to proteins, subunits,domains, motifs, and/or epitopes belonging to the target CD19.

Fc Variants and Fc Receptor Binding Properties

Immunoglobulins disclosed herein comprise an Fc variant. An Fc variantcomprises one or more amino acid modifications relative to a parent Fcpolypeptide, wherein the amino acid modification(s) provide one or moreoptimized properties. An Fc variant disclosed herein differs in aminoacid sequence from its parent by virtue of at least one amino acidmodification. Thus Fc variants disclosed herein have at least one aminoacid modification compared to the parent. Alternatively, the Fc variantsdisclosed herein may have more than one amino acid modification ascompared to the parent, for example from about two to fifty amino acidmodifications, e.g., from about two to ten amino acid modifications,from about two to about five amino acid modifications, etc. compared tothe parent. Thus the sequences of the Fc variants and those of theparent Fc polypeptide are substantially homologous. For example, thevariant Fc variant sequences herein will possess about 80% homology withthe parent Fc variant sequence, e.g., at least about 90% homology, atleast about 95% homology, at least about 98% homology, at least about99% homology, etc. Modifications disclosed herein include amino acidmodifications, including insertions, deletions, and substitutions.Modifications disclosed herein also include glycoform modifications.Modifications may be made genetically using molecular biology, or may bemade enzymatically or chemically.

Fc variants disclosed herein are defined according to the amino acidmodifications that compose them. Thus, for example, S267E is an Fcvariant with the substitution S267E relative to the parent Fcpolypeptide. Likewise, S267E/L328F defines an Fc variant with thesubstitutions S267E and L328F relative to the parent Fc polypeptide. Theidentity of the WT amino acid may be unspecified, in which case theaforementioned variant is referred to as 267E/328F. It is noted that theorder in which substitutions are provided is arbitrary, that is to saythat, for example, 267E/328F is the same Fc variant as 328F/267E, and soon. Unless otherwise noted, positions discussed herein are numberedaccording to the EU index as described in Kabat (Kabat et al., 1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda, herebyentirely incorporated by reference). In brief, EU is the name of thefirst antibody molecule whose entire amino acid sequence was determined(Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirelyincorporated by reference), and its amino acid sequence has become thestandard numbering scheme for heavy chain constant regions. The EUprotein has become the standard reference for defining numbering. Kabatet al. lists the EU sequence in a set of indices aligning it with otherantibody sequences, serving as a necessary tool for aligning antibodiesto the EU numbering scheme. Thus, as appreciated by those of skill inthe art, the standard way of referencing the EU numbering is to refer toKabat et al.'s alignment of sequences, because it puts EU in contextwith antibodies of other variable domain lengths. As such, as usedherein, “the EU index as in Kabat” or “numbering is according to the EUindex, as in Kabat” refers to the numbering of the EU antibody asdescribed in Kabat.

In certain embodiments, the Fc variants disclosed herein are based onhuman IgG sequences, and thus human IgG sequences are used as the “base”sequences against which other sequences are compared, including but notlimited to sequences from other organisms, for example rodent andprimate sequences. Immunoglobulins may also comprise sequences fromother immunoglobulin classes such as IgA, IgE, IgGD, IgGM, and the like.It is contemplated that, although the Fc variants disclosed herein areengineered in the context of one parent IgG, the variants may beengineered in or “transferred” to the context of another, second parentIgG. This is done by determining the “equivalent” or “corresponding”residues and substitutions between the first and second IgG, typicallybased on sequence or structural homology between the sequences of thefirst and second IgGs. In order to establish homology, the amino acidsequence of a first IgG outlined herein is directly compared to thesequence of a second IgG. After aligning the sequences, using one ormore of the homology alignment programs known in the art (for exampleusing conserved residues as between species), allowing for necessaryinsertions and deletions in order to maintain alignment (i.e., avoidingthe elimination of conserved residues through arbitrary deletion andinsertion), the residues equivalent to particular amino acids in theprimary sequence of the first immunoglobulin are defined. Alignment ofconserved residues may conserve 100% of such residues. However,alignment of greater than 75% or as little as 50% of conserved residuesis also adequate to define equivalent residues. Equivalent residues mayalso be defined by determining structural homology between a first andsecond IgG that is at the level of tertiary structure for IgGs whosestructures have been determined. In this case, equivalent residues aredefined as those for which the atomic coordinates of two or more of themain chain atoms of a particular amino acid residue of the parent orprecursor (N on N, CA on CA, C on C and 0 on 0) are within about 0.13nm, after alignment. In another embodiment, equivalent residues arewithin about 0.1 nm after alignment. Alignment is achieved after thebest model has been oriented and positioned to give the maximum overlapof atomic coordinates of non-hydrogen protein atoms of the proteins.Regardless of how equivalent or corresponding residues are determined,and regardless of the identity of the parent IgG in which the IgGs aremade, what is meant to be conveyed is that the Fc variants discovered asdisclosed herein may be engineered into any second parent IgG that hassignificant sequence or structural homology with the Fc variant. Thusfor example, if a variant antibody is generated wherein the parentantibody is human IgG1, by using the methods described above or othermethods for determining equivalent residues, the variant antibody may beengineered in another IgG1 parent antibody that binds a differentantigen, a human IgG2 parent antibody, a human IgA parent antibody, amouse IgG2a or IgG2b parent antibody, and the like. Again, as describedabove, the context of the parent Fc variant does not affect the abilityto transfer the Fc variants disclosed herein to other parent IgGs.

The Fc variants disclosed herein may be optimized for a variety of Fcreceptor binding properties. An Fc variant that is engineered orpredicted to display one or more optimized properties is herein referredto as an “optimized Fc variant.” Properties that may be optimizedinclude but are not limited to enhanced or reduced affinity for an FcγR.In one embodiment, the Fc variants disclosed herein are optimized topossess enhanced affinity for an inhibitory receptor FcγRIIb. In otherembodiments, immunoglobulins disclosed herein provide enhanced affinityfor FcγRIIb, yet reduced affinity for one or more activating FcγRs,including for example FcγRI, FcγRIIa, FcγRIIIa, and/or FcγRIIIb. TheFcγR receptors may be expressed on cells from any organism, includingbut not limited to human, cynomolgus monkeys, and mice. The Fc variantsdisclosed herein may be optimized to possess enhanced affinity for humanFcγRIIb.

By “greater affinity” or “improved affinity” or “enhanced affinity” or“better affinity” than a parent Fc polypeptide, as used herein is meantthat an Fc variant binds to an Fc receptor with a significantly higherequilibrium constant of association (K_(A) or Ka) or lower equilibriumconstant of dissociation (K_(D) or Kd) than the parent Fc polypeptidewhen the amounts of variant and parent polypeptide in the binding assayare essentially the same. For example, the Fc variant with improved Fcreceptor binding affinity may display from about 5 fold to about 1000fold, e.g. from about 10 fold to about 500 fold improvement in Fcreceptor binding affinity compared to the parent Fc polypeptide, whereFc receptor binding affinity is determined, for example, by the bindingmethods disclosed herein, including but not limited to Biacore, by oneskilled in the art. Accordingly, by “reduced affinity” as compared to aparent Fc polypeptide as used herein is meant that an Fc variant bindsan Fc receptor with significantly lower K_(A) or higher K_(D) than theparent Fc polypeptide. Greater or reduced affinity can also be definedrelative to an absolute level of affinity. For example, according to thedata herein, WT (native) IgG1 binds FcγRIIb with an affinity of about1.5 μM or about 1500 nM. Furthermore, some Fc variants described hereinbind FcγRIIb with an affinity about 10-fold greater to WT IgG1. Asdisclosed herein, greater or enhanced affinity means having a K_(D)lower than about 100 nM, for example between about 10 nM-about 100 nM,between about 1-about 100 nM, or less than about 1 nM.

In one embodiment, the Fc variants provide selectively enhanced affinityto FcγRIIb relative to one or more activating receptors. Selectivelyenhanced affinity means either that the Fc variant has improved affinityfor FcγRIIb relative to the activating receptor(s) as compared to theparent Fc polypeptide but has reduced affinity for the activatingreceptor(s) as compared to the parent Fc polypeptide, or it means thatthe Fc variant has improved affinity for both FcγRIIb and activatingreceptor(s) as compared to the parent Fc polypeptide, however theimprovement in affinity is greater for FcγRIIb than it is for theactivating receptor(s). In alternate embodiments, the Fc variants reduceor ablate binding to one or more activating FcγRs, reduce or ablatebinding to one or more complement proteins, reduce or ablate one or moreFcγR-mediated effector functions, and/or reduce or ablate one or morecomplement-mediated effector functions.

The presence of different polymorphic forms of FcγRs provides yetanother parameter that impacts the therapeutic utility of the Fcvariants disclosed herein. Whereas the specificity and selectivity of agiven Fc variant for the different classes of FcγRs significantlyaffects the capacity of an Fc variant to target a given antigen fortreatment of a given disease, the specificity or selectivity of an Fcvariant for different polymorphic forms of these receptors may in partdetermine which research or pre-clinical experiments may be appropriatefor testing, and ultimately which patient populations may or may notrespond to treatment. Thus the specificity or selectivity of Fc variantsdisclosed herein to Fc receptor polymorphisms, including but not limitedto FcγRIIa, FcγRIIIa, and the like, may be used to guide the selectionof valid research and pre-clinical experiments, clinical trial design,patient selection, dosing dependence, and/or other aspects concerningclinical trials.

Fc variants disclosed herein may comprise modifications that modulateinteraction with Fc receptors other than FcγRs, including but notlimited to complement proteins, FcRn, and Fc receptor homologs (FcRHs).FcRHs include but are not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5,and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136).

An important parameter that determines the most beneficial selectivityof a given Fc variant to treat a given disease is the context of the Fcvariant. Thus the Fc receptor selectivity or specificity of a given Fcvariant will provide different properties depending on whether itcomposes an antibody, Fc fusion, or Fc variants with a coupled fusionpartner. In one embodiment, an Fc receptor specificity of the Fc variantdisclosed herein will determine its therapeutic utility. The utility ofa given Fc variant for therapeutic purposes will depend on the epitopeor form of the target antigen and the disease or indication beingtreated. For some targets and indications, greater FcγRIIb affinity andreduced activating FcγR-mediated effector functions may be beneficial.For other target antigens and therapeutic applications, it may bebeneficial to increase affinity for FcγRIIb, or increase affinity forboth FcγRIIb and activating receptors.

Inhibitory Properties and Methods of Inhibiting CD32b⁺ Cells

The target antigen (CD19) of immunoglobulins disclosed herein may beexpressed on a variety of cell types. In some embodiments,immunoglobulins disclosed herein are specific for CD19 expressed onCD32b+ cells. Cell types that may be targeted by the immunoglobulinsdisclosed herein include, but are not limited to, B cells, plasma cells,dendritic cells, macrophages, neutrophils, mast cells, basophils, andeosinophils.

Disclosed herein are methods of inhibiting CD32b+ cells. Without beinglimited thereto, FIG. 3 is a schematic representation of a proposedmechanism by which immunoglobulins disclosed herein inhibit CD32b+cells. Accordingly, disclosed herein are methods of inhbiting CD32b+cells comprising contacting a CD32b+ cell with an immunoglobulincomprising an Fc region with enhanced affinity to FcγRIIb. Theimmunoglobulin binds at least two B cell proteins, e.g., at least twoproteins bound to the surface B cells. In one embodiment, the first ofsaid B cell proteins is FcγRIIb. In another embodiment, the second ofsaid B cell proteins is CD19. In some embodiments, the immunoglobulinsinhibit release of calcium from the B cells upon their stimulationthrough the B cell receptor. In another embodiment, an immunoglobulindisclosed herein binds at least two B cell proteins on the surface ofthe same B cell (see, e.g., FIG. 3).

Modifications for Optimizing Inhibitory Function

Disclosed herein is directed to immunoglobulins comprisingmodifications, wherein said modifications alter affinity to the FcγRIIbreceptor, and/or alter the ability of the immunoglobulin to mediate oneor more FcγRIIb-mediated effector functions. Modifications of thedisclosure include amino acid modifications and glycoform modifications.

As described herein, simultaneous high affinity coengagement of cognateBCR and FcγRIIb may be used to inhibit FcγRIIb+ cells. Such coengagmentmay occur via the use of an immunoglobulin described herein, e.g., animmunoglobulin used to coengage both FcγRIIb via its Fc region, and atarget antigen on the surface of the FcγRIIb+ cell (e.g., CD19) via itsFv region. Amino acid modifications at heavy chain constant regionpositions 234, 235, 236, 239, 267, 268, and 328, allow modification ofimmunoglobulin FcγRIIb binding properties, effector function, andpotentially clinical properties of antibodies.

In one embodiment, immunoglobulins that bind FcγRIIb+ cells and coengagea target antigen on the cell's surface and an FcγRIIb on cell's surfacedisclosed herein may be variant immunoglobulins relative to a parentimmunoglobulin. In one embodiment, the variant immunoglobulin comprisesa variant Fc region, wherein said variant Fc region comprises one ormore (e.g., two or more) modifications(s) compared to a parent Fcregions, wherein said modification(s) are at positions selected from thegroup consisting of 234, 235, 236, 239, 267, 268, and 328, whereinnumbering is according to an EU index as in Kabat. In one embodiment,wherein said variant Fc region comprises one or more (e.g., two or more)modifications(s) compared to a parent Fc regions, wherein saidmodification(s) are at positions selected from the group consisting of267 and 328 according to the EU index as in Kabat.

In one embodiment, said modification(s) is at least one substitution(e.g., one or more substitution(s), two or more substitution(s), etc.)selected from the group consisting of 234W, 235I, 235Y, 235R, 235D,236D, 236N, 239D, 267D, 267E, 268E, 268D, 328F, and 328Y, whereinnumbering is according to an EU index as in Kabat. In anotherembodiment, said modification(s) is at least one substitution selectedfrom the group consisting of 235Y, 235R, 236D, 267D, 267E, and 328F. Inone embodiment, said modification(s) is at least one substitutionselected from the group consisting of 267E and 328F, wherein numberingis according to an EU index as in Kabat. In one embodiment, saidmodification(s) is at least one substitution selected from the groupconsisting of L234W, L235I, L235Y, L235R, L235D, G236D, G236N, S239D,S267D, S267E, H268E, H268D, L328F, and L328Y wherein numbering isaccording to an EU index as in Kabat. In another embodiment, saidmodification(s) is at least one substitution selected from the groupconsisting of L235Y, L235R, G236D, S267D, S267E, and L328F. In oneembodiment, said modification(s) is at least one substitution selectedfrom the group consisting of S267E and L328F, wherein numbering isaccording to an EU index as in Kabat.

In another embodiment, said modification(s) is at least twomodifications (e.g., a combination of modifications) at positionsselected from the group consisting of 235/267, 236/267, 236/267,267/328, and 268/267, wherein numbering is according to an EU index asin Kabat. In another embodiment, said modification(s) is at least twosubstitutions at positions 267/328, wherein numbering is according to anEU index as in Kabat. In a further embodiment, said modification(s) isat least two substitutions selected from the group consisting of235D/267E, 235Y/267E, 235D/S267D, 235I/267E, 235I/267D, 235Y/267D,236D/267E, 236D/267D, 267E/328F, 267D/328F, 268D/267E, 268D/267D,268E/267E, and 268E/267D, wherein numbering is according to an EU indexas in Kabat. In another embodiment, said modification(s) is at least twosubstitutions selected from the group consisting of 235D/267E,235Y/267E, 235Y/267D, 236D/267E, 267E/328F, 268D/267E, 268E/267E, and268E/267D, wherein numbering is according to an EU index as in Kabat. Inone embodiment, said modification(s) is at least 267E/328F, whereinnumbering is according to an EU index as in Kabat. In anotherembodiment, said modification(s) is at least two substitutions selectedfrom the group consisting of L235D/S267E, L235Y/S267E, L235D/S267D,L235I/S267E, L235I/S267D, L235Y/S267D, G236D/S267E, G236D/S267D,S267E/L328F, S267D/L328F, H268D/S267E, H268D/S267D, H268E/S267E, andH268E/S267D, wherein numbering is according to an EU index as in Kabat.In another embodiment, said modification(s) is at least twosubstitutions selected from the group consisting of L235D/S267E,L235Y/S267E, L235Y/S267D, G236D/S267E, S267E/L328F, H268D/S267E,H268E/S267E, and H268E/S267D, wherein numbering is according to an EUindex as in Kabat. In one embodiment, said modification(s) is at leastS267E/L328F, wherein numbering is according to an EU index as in Kabat.

In another embodiment, said modification(s) is at least threesubstitutions (e.g., a combination of modifications) at positions236/267/328, wherein numbering is according to an EU index as in Kabat.In another embodiment, said modification(s) is at least 236D/267E/328F.In another embodiment, said substitutional means is at leastG236D/S267E/L328F.

In some embodiments, antibodies may comprise isotypic modifications,that is, modifications in a parent IgG to the amino acid type in analternate IgG. For example as illustrated in FIG. 1, an IgG1/IgG3 hybridvariant may be constructed by substituting IgG1 positions in the CH2and/or CH3 region with the amino acids from IgG3 at positions where thetwo isotypes differ. Thus a hybrid variant IgG antibody may beconstructed that comprises one or more substitutions selected from thegroup consisting of: 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N,397M, 4221, 435R, and 436F. In other embodiments of the disclosure, anIgG1/IgG2 hybrid variant may be constructed by substituting IgG2positions in the CH2 and/or CH3 region with amino acids from IgG1 atpositions where the two isotypes differ. Thus a hybrid variant IgGantibody may be constructed that comprises one or more modificationsselected from the group consisting of 233E, 234L, 235L, -236G (referringto an insertion of a glycine at position 236), and 327A.

Means for Optimizing Effector Function

Described herein are immunoglobulins comprising means for alteringaffinity to the FcγRIIb receptor, and/or altering the ability of theimmunoglobulin to mediate one or more FcγRIIb-mediated effectorfunctions. Means of the disclosure include amino acid modifications(e.g., positional means for optimizing effector function, substitutionalmeans for optimizing effector function, etc.) and glycoformmodifications (e.g., means for glycoform modifications).

Amino Acid Modifications

As described herein, positional means for optimizing effector functioninclude, but is not limited to, modification of an amino acid at one ormore heavy chain constant region positions 234, 235, 236, 239, 267, 268,and 328, which allow modification of immunoglobulin FcγRIIb bindingproperties, effector function, and potentially clinical properties ofantibodies.

In particular, substitutional means for optimizing FcγRIIb effectorfunctions, e.g., by altering affinity to FcγRIIb, include, but is notlimited to, a substitution of an amino acid at one or more heavy chainconstant region positions, e.g., one or more of the amino acidsubstitutions in the heavy chain constant region positions 234, 235,236, 239, 267, 268, and 328, wherein numbering is according to an EUindex as in Kabat. In one embodiment, substitutional means include atleast one substitution(s) (e.g., one or more substitution(s), two ormore substitution(s), etc.) compared to a parent Fc region, wherein saidsubstitution(s) are at positions selected from the group consisting of267 and 328 according to the EU index as in Kabat. In one embodiment,said substitutional means is at least one substitution selected from thegroup consisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D,267D, 267E, 268E, 268D, 328F, and 328Y, wherein numbering is accordingto an EU index as in Kabat. In another embodiment, said substitutionalmeans is at least one substitution selected from the group consisting of235Y, 235R, 236D, 267D, 267E, and 328F, wherein numbering is accordingto an EU index as in Kabat. In one embodiment, said substitutional meansis at least one substitution selected from the group consisting of 267Eand 328F, wherein numbering is according to an EU index as in Kabat. Inone embodiment, said substitutional means is at least one substitutionselected from the group consisting of L234W, L235I, L235Y, L235R, L235D,G236D, G236N, S239D, S267D, S267E, H268E, H268D, L328F, and L328Y,wherein numbering is according to an EU index as in Kabat. In anotherembodiment, said substitutional means is at least one substitutionselected from the group consisting of L235Y, L235R, G236D, S267D, S267E,and L328F, wherein numbering is according to an EU index as in Kabat. Inone embodiment, said substitutional means is at least one substitutionselected from the group consisting of S267E and L328F, wherein numberingis according to an EU index as in Kabat.

In another embodiment, said substitutional means is at least twosubstitutions (e.g., a combination of modifications) at positions235/267, 236/267, 236/267, 267/328, and 268/267, wherein numbering isaccording to an EU index as in Kabat. In another embodiment, saidsubstitutional means is at least two substitutions at positions 267/328,wherein numbering is according to an EU index as in Kabat. In a furtherembodiment, said substitutional means is at least two substitutions atpositions 235D/267E, 235Y/267E, 235D/S267D, 235I/267E, 235I/267D,235Y/267D, 236D/267E, 236D/267D, 267E/328F, 267D/328F, 268D/267E,268D/267D, 268E/267E, and 268E/267D, wherein numbering is according toan EU index as in Kabat. In another embodiment, said substitutionalmeans is at least two substitutions at positions 235D/267E, 235Y/267E,235Y/267D, 236D/267E, 267E/328F, 268D/267E, 268E/267E, and 268E/267D,wherein numbering is according to an EU index as in Kabat. In oneembodiment, said substitutional means is at least 267E/328F, whereinnumbering is according to an EU index as in Kabat. In anotherembodiment, said substitutional means is at least two substitutions atpositions L235D/S267E, L235Y/S267E, L235D/S267D, L235I/S267E,L235I/S267D, L235Y/S267D, G236D/S267E, G236D/S267D, S267E/L328F,S267D/L328F, H268D/S267E, H268D/S267D, H268E/S267E, and H268E/S267D,wherein numbering is according to an EU index as in Kabat. In anotherembodiment, said substitutional means is at least two substitutions atpositions L235D/S267E, L235Y/S267E, L235Y/S267D, G236D/S267E,S267E/L328F, H268D/S267E, H268E/S267E, and H268E/S267D, whereinnumbering is according to an EU index as in Kabat. In one embodiment,said substitutional means is at least S267E/L328F, wherein numbering isaccording to an EU index as in Kabat.

In another embodiment, said substitutional means is at least threesubstitutions (e.g., a combination of modifications) at positions236/267/328, wherein numbering is according to an EU index as in Kabat.In another embodiment, said substitutional means is at least236D/267E/328F, wherein numbering is according to an EU index as inKabat. In another embodiment, said substitutional means is at leastG236D/S267E/L328F, wherein numbering is according to an EU index as inKabat.

Glycoform Modifications

Many polypeptides, including antibodies, are subjected to a variety ofpost-translational modifications involving carbohydrate moieties, suchas glycosylation with oligosaccharides. There are several factors thatcan influence glycosylation. The species, tissue and cell type have allbeen shown to be important in the way that glycosylation occurs. Inaddition, the extracellular environment, through altered cultureconditions such as serum concentration, may have a direct effect onglycosylation (Lifely et al., 1995, Glycobiology 5(8): 813-822).

All antibodies contain carbohydrate at conserved positions in theconstant regions of the heavy chain. Each antibody isotype has adistinct variety of N-linked carbohydrate structures. Aside from thecarbohydrate attached to the heavy chain, up to 30% of human IgGs have aglycosylated Fab region. IgG has a single N-linked biantennarycarbohydrate at Asn297 of the CH2 domain. For IgG from either serum orproduced ex vivo in hybridomas or engineered cells, the IgG areheterogeneous with respect to the Asn297 linked carbohydrate (Jefferiset al., 1998, Immunol. Rev. 163:59-76; Wright et al., 1997, TrendsBiotech 15:26-32). For human IgG, the core oligosaccharide normallyconsists of GlcNAc₂Man₃GlcNAc, with differing numbers of outer residues.

The carbohydrate moieties of immunoglobulins disclosed herein will bedescribed with reference to commonly used nomenclature for thedescription of oligosaccharides. A review of carbohydrate chemistrywhich uses this nomenclature is found in Hubbard et al. 1981, Ann. Rev.Biochem. 50:555-583. This nomenclature includes, for instance, Man,which represents mannose; GlcNAc, which represents2-N-acetylglucosamine; Gal which represents galactose; Fuc for fucose;and Glc, which represents glucose. Sialic acids are described by theshorthand notation NeuNAc, for 5-N-acetylneuraminic acid, and NeuNGc for5-glycolylneuraminic.

The term “glycosylation” means the attachment of oligosaccharides(carbohydrates containing two or more simple sugars linked together e.g.from two to about twelve simple sugars linked together) to aglycoprotein. The oligosaccharide side chains are typically linked tothe backbone of the glycoprotein through either N- or O-linkages. Theoligosaccharides of immunoglobulins disclosed herein occur generally areattached to a CH2 domain of an Fc region as N-linked oligosaccharides.“N-linked glycosylation” refers to the attachment of the carbohydratemoiety to an asparagine residue in a glycoprotein chain. The skilledartisan will recognize that, for example, each of murine IgG1, IgG2a,IgG2b and IgG3 as well as human IgG1, IgG2, IgG3, IgG4, IgA and IgD CH2domains have a single site for N-linked glycosylation at amino acidresidue 297 (Kabat et al. Sequences of Proteins of ImmunologicalInterest, 1991).

For the purposes herein, a “mature core carbohydrate structure” refersto a processed core carbohydrate structure attached to an Fc regionwhich generally consists of the following carbohydrate structureGlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc)₂ typical of biantennaryoligosaccharides. The mature core carbohydrate structure is attached tothe Fc region of the glycoprotein, generally via N-linkage to Asn297 ofa CH2 domain of the Fc region. A “bisecting GlcNAc” is a GlcNAc residueattached to the β1,4 mannose of the mature core carbohydrate structure.The bisecting GlcNAc can be enzymatically attached to the mature corecarbohydrate structure by a β(1,4)-N-acetylglucosaminyltransferase IIIenzyme (GnTIII). CHO cells do not normally express GnTIII (Stanley etal., 1984, J. Biol. Chem. 261:13370-13378), but may be engineered to doso (Umana et al., 1999, Nature Biotech. 17:176-180).

Described herein are Fc variants that comprise modified glycoforms orengineered glycoforms. By “modified glycoform” or “engineered glycoform”as used herein is meant a carbohydrate composition that is covalentlyattached to a protein, for example an antibody, wherein saidcarbohydrate composition differs chemically from that of a parentprotein. Engineered glycoforms may be useful for a variety of purposes,including but not limited to enhancing or reducing FcγR-mediatedeffector function. In one embodiment, the immunoglobulins disclosedherein are modified to control the level of fucosylated and/or bisectingoligosaccharides that are covalently attached to the Fc region.

A variety of methods are well known in the art for generating modifiedglycoforms (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies etal., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473);(U.S. Pat. No. 6,602,684; US Pub. No. 2003/0157108; US Pub. No.2003/0003097; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1;PCT WO 02/30954A1); (Potelligent™ technology [Biowa, Inc., Princeton,N.J.]; GlycoMAb™ glycosylation engineering technology [GLYCARTbiotechnology AG, Zurich, Switzerland]; all of which are expresslyincorporated by reference). These techniques control the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells), by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed. Othermethods for modifying glycoforms of the immunoglobulins disclosed hereininclude using glycoengineered strains of yeast (Li et al., 2006, NatureBiotechnology 24(2):210-215), moss (Nechansky et al., 2007, Mol Immunjol44(7):1826-8), and plants (Cox et al., 2006, Nat Biotechnol24(12):1591-7). The use of a particular method to generate a modifiedglycoform is not meant to constrain embodiments to that method. Rather,embodiments disclosed herein encompass Fc variants with modifiedglycoforms irrespective of how they are produced.

In one embodiment, immunoglobulins disclosed herein are glycoengineeredto alter the level of sialylation. Higher levels of sialylated Fcglycans in immunoglobulin G molecules can adversely impact functionality(Scallon et al., 2007, Mol Immunol. 44(7):1524-34), and differences inlevels of Fc sialylation can result in modified anti-inflammatoryactivity (Kaneko et al., 2006, Science 313:670-673). Because antibodiesmay acquire anti-inflammatory properties upon sialylation of Fc corepolysaccharide, it may be advantageous to glycoengineer theimmunoglobulins disclosed herein for greater or reduced Fc sialic acidcontent.

Engineered glycoform typically refers to the different carbohydrate oroligosaccharide; thus for example an immunoglobulin may comprise anengineered glycoform. Alternatively, engineered glycoform may refer tothe immunoglobulin that comprises the different carbohydrate oroligosaccharide. In one embodiment, a composition disclosed hereincomprises a glycosylated Fc variant having an Fc region, wherein about51-100% of the glycosylated antibody, e.g., 80-100%, 90-100%, 95-100%,etc. of the antibody in the composition comprises a mature corecarbohydrate structure which lacks fucose. In another embodiment, theantibody in the composition both comprises a mature core carbohydratestructure that lacks fucose and additionally comprises at least oneamino acid modification in the Fc region. In an alternative embodiment,a composition comprises a glycosylated Fc variant having an Fc region,wherein about 51-100% of the glycosylated antibody, 80-100%, or 90-100%,of the antibody in the composition comprises a mature core carbohydratestructure which lacks sialic acid. In another embodiment, the antibodyin the composition both comprises a mature core carbohydrate structurethat lacks sialic acid and additionally comprises at least one aminoacid modification in the Fc region. In yet another embodiment, acomposition comprises a glycosylated Fc variant having an Fc region,wherein about 51-100% of the glycosylated antibody, 80-100%, or 90-100%,of the antibody in the composition comprises a mature core carbohydratestructure which contains sialic acid. In another embodiment, theantibody in the composition both comprises a mature core carbohydratestructure that contains sialic acid and additionally comprises at leastone amino acid modification in the Fc region. In another embodiment, thecombination of engineered glycoform and amino acid modification providesoptimal Fc receptor binding properties to the antibody.

Other Modifications

Immunoglobulins disclosed herein may comprise one or more modificationsthat provide optimized properties that are not specifically related toFcγR- or complement-mediated effector functions per se. Saidmodifications may be amino acid modifications, or may be modificationsthat are made enzymatically or chemically. Such modification(s) likelyprovide some improvement in the immunoglobulin, for example anenhancement in its stability, solubility, function, or clinical use.Disclosed herein are a variety of improvements that may be made bycoupling the immunoglobulins disclosed herein with additionalmodifications.

In one embodiment, the variable region of an antibody disclosed hereinmay be affinity matured, that is to say that amino acid modificationshave been made in the VH and/or VL domains of the antibody to enhancebinding of the antibody to its target antigen. Such types ofmodifications may improve the association and/or the dissociationkinetics for binding to the target antigen. Other modifications includethose that improve selectivity for target antigen vs. alternativetargets. These include modifications that improve selectivity forantigen expressed on target vs. non-target cells. Other improvements tothe target recognition properties may be provided by additionalmodifications. Such properties may include, but are not limited to,specific kinetic properties (i.e. association and dissociationkinetics), selectivity for the particular target versus alternativetargets, and selectivity for a specific form of target versusalternative forms. Examples include full-length versus splice variants,cell-surface vs. soluble forms, selectivity for various polymorphicvariants, or selectivity for specific conformational forms of the targetantigen. Immunoglobulins disclosed herein may comprise one or moremodifications that provide reduced or enhanced internalization of animmunoglobulin.

In one embodiment, modifications are made to improve biophysicalproperties of the immunoglobulins disclosed herein, including, but notlimited to stability, solubility, and oligomeric state. Modificationscan include, for example, substitutions that provide more favorableintramolecular interactions in the immunoglobulin such as to providegreater stability, or substitution of exposed nonpolar amino acids withpolar amino acids for higher solubility. A number of optimization goalsand methods are described in U.S. Patent Publication No. 2004/0110226,incorporated entirely by reference, that may find use for engineeringadditional modifications to further optimize the immunoglobulinsdisclosed herein. The immunoglobulins disclosed herein can also becombined with additional modifications that reduce oligomeric state orsize, such that tumor penetration is enhanced, or in vivo clearancerates are increased as desired.

Other modifications to the immunoglobulins disclosed herein includethose that enable the specific formation or homodimeric orhomomultimeric molecules. Such modifications include but are not limitedto engineered disulfides, as well as chemical modifications oraggregation methods which may provide a mechanism for generatingcovalent homodimeric or homomultimers. For example, methods ofengineering and compositions of such molecules are described in Kan etal., 2001, J. Immunol., 2001, 166: 1320-1326; Stevenson et al., 2002,Recent Results Cancer Res. 159: 104-12; U.S. Pat. No. 5,681,566; Caronet al., 1992, J. Exp. Med. 176:1191-1195, and Shopes, 1992, J. Immunol.148(9):2918-22, all incorporated entirely by reference. Additionalmodifications to the variants disclosed herein include those that enablethe specific formation or heterodimeric, heteromultimeric, bifunctional,and/or multifunctional molecules. Such modifications include, but arenot limited to, one or more amino acid substitutions in the CH3 domain,in which the substitutions reduce homodimer formation and increaseheterodimer formation. For example, methods of engineering andcompositions of such molecules are described in Atwell et al., 1997, J.Mol. Biol. 270(1):26-35, and Carter et al., 2001, J. Immunol. Methods248:7-15, both incorporated entirely by reference. Additionalmodifications include modifications in the hinge and CH3 domains, inwhich the modifications reduce the propensity to form dimers.

In further embodiments, the immunoglobulins disclosed herein comprisemodifications that remove proteolytic degradation sites. These mayinclude, for example, protease sites that reduce production yields, aswell as protease sites that degrade the administered protein in vivo. Inone embodiment, additional modifications are made to remove covalentdegradation sites such as deamidation (i.e. deamidation of glutaminyland asparaginyl residues to the corresponding glutamyl and aspartylresidues), oxidation, and proteolytic degradation sites. Deamidationsites that are particular useful to remove are those that have enhancepropensity for deamidation, including, but not limited to asparaginyland gltuamyl residues followed by glycines (NG and QG motifs,respectively). In such cases, substitution of either residue cansignificantly reduce the tendency for deamidation. Common oxidationsites include methionine and cysteine residues. Other covalentmodifications, that can either be introduced or removed, includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the “-amino groups oflysine, arginine, and histidine side chains (T. E. Creighton, Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,pp. 79-86 (1983), incorporated entirely by reference), acetylation ofthe N-terminal amine, and amidation of any C-terminal carboxyl group.Additional modifications also may include but are not limited toposttranslational modifications such as N-linked or O-linkedglycosylation and phosphorylation.

Modifications may include those that improve expression and/orpurification yields from hosts or host cells commonly used forproduction of biologics. These include, but are not limited to variousmammalian cell lines (e.g. CHO), yeast cell lines, bacterial cell lines,and plants. Additional modifications include modifications that removeor reduce the ability of heavy chains to form inter-chain disulfidelinkages. Additional modifications include modifications that remove orreduce the ability of heavy chains to form intra-chain disulfidelinkages.

The immunoglobulins disclosed herein may comprise modifications thatinclude the use of unnatural amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci.U.S.A. 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin etal., 2003, Science 301(5635):964-7, all incorporated entirely byreference. In some embodiments, these modifications enable manipulationof various functional, biophysical, immunological, or manufacturingproperties discussed above. In additional embodiments, thesemodifications enable additional chemical modification for otherpurposes. Other modifications are contemplated herein. For example, theimmunoglobulin may be linked to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol. Additional amino acid modifications may be made to enablespecific or non-specific chemical or posttranslational modification ofthe immunoglobulins. Such modifications, include, but are not limited toPEGylation and glycosylation. Specific substitutions that can beutilized to enable PEGylation include, but are not limited to,introduction of novel cysteine residues or unnatural amino acids suchthat efficient and specific coupling chemistries can be used to attach aPEG or otherwise polymeric moiety. Introduction of specificglycosylation sites can be achieved by introducing novel N-X-T/Ssequences into the immunoglobulins disclosed herein.

Modifications to reduce immunogenicity may include modifications thatreduce binding of processed peptides derived from the parent sequence toMHC proteins. For example, amino acid modifications would be engineeredsuch that there are no or a minimal number of immune epitopes that arepredicted to bind, with high affinity, to any prevalent MHC alleles.Several methods of identifying MHC-binding epitopes in protein sequencesare known in the art and may be used to score epitopes in an antibodydisclosed herein. See for example U.S. Patent Publication Nos.2002/0119492, 2004/0230380, 2006/0148009, and references cited therein,all expressly incorporated by reference.

In some embodiments, immunoglobulins disclosed herein may be combinedwith immunoglobulins that alter FcRn binding. Such variants may provideimproved pharmacokinetic properties to the immunoglobulins. Inparticular, variants that increase Fc binding to FcRn include but arenot limited to: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004,J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal ofImmunology 176:346-356, U.S. Ser. No. 11/102,621, PCT/US2003/033037,PCT/US2004/011213, U.S. Ser. No. 10/822,300, U.S. Ser. No. 10/687,118,PCT/US2004/034440, U.S. Ser. No. 10/966,673 all entirely incorporated byreference), 256A, 272A, 286A, 305A, 307A, 311A, 312A, 376A, 378Q, 380A,382A, 434A (Shields et al, Journal of Biological Chemistry, 2001,276(9):6591-6604, U.S. Ser. No. 10/982,470, U.S. Pat. No. 6,737,056,U.S. Ser. No. 11/429,793, U.S. Ser. No. 11/429,786, PCT/US2005/029511,U.S. Ser. No. 11/208,422, all entirely incorporated by reference), 252F,252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S,433R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y/254T/256E,433K/434F/436H, 308T/309P/311 S (DaII Acqua et al. Journal ofImmunology, 2002, 169:5171-5180, U.S. Pat. No. 7,083,784,PCT/US97/03321, U.S. Pat. No. 6,821,505, PCT/US01/48432, U.S. Ser. No.11/397,328, all entirely incorporated by reference), 257C, 257M, 257L,257N, 257Y, 279E, 279Q, 279Y, insertion of Ser after 281, 283F, 284E,306Y, 307V, 308F, 308Y 311V, 385H, 385N, (PCT/US2005/041220, U.S. Ser.No. 11/274,065, U.S. Ser. No. 11/436,266 all entirely incorporated byreference) 204D, 284E, 285E, 286D, and 290E (PCT/US2004/037929 entirelyincorporated by reference).

Covalent modifications of antibodies are included within the scope ofimmunoglobulins disclosed herein, and are generally, but not always,done post-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

In some embodiments, the covalent modification of the antibodiesdisclosed herein comprises the addition of one or more labels. The term“labeling group” means any detectable label. In some embodiments, thelabeling group is coupled to the antibody via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used in generatingimmunoglobulins disclosed herein. In general, labels fall into a varietyof classes, depending on the assay in which they are to be detected: a)isotopic labels, which may be radioactive or heavy isotopes; b) magneticlabels (e.g., magnetic particles); c) redox active moieties; d) opticaldyes; enzymatic groups (e.g. horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase); e) biotinylated groups; and f)predetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags, etc.). In someembodiments, the labeling group is coupled to the antibody via spacerarms of various lengths to reduce potential steric hindrance. Variousmethods for labeling proteins are known in the art and may be used ingenerating immunoglobulins disclosed herein. Specific labels includeoptical dyes, including, but not limited to, chromophores, phosphors,and fluorophores, with the latter being specific in many instances.Fluorophores can be either “small molecule” fluores, or proteinaceousfluores. By “fluorescent label” is meant any molecule that may bedetected via its inherent fluorescent properties.

Conjugates

In one embodiment, the immunoglobulins disclosed herein are antibody“fusion proteins,” sometimes referred to herein as “antibodyconjugates.” The fusion partner or conjugate partner can beproteinaceous or non-proteinaceous; the latter generally being generatedusing functional groups on the antibody and on the conjugate partner.Conjugate and fusion partners may be any molecule, including smallmolecule chemical compounds and polypeptides. For example, a variety ofantibody conjugates and methods are described in Trail et al., 1999,Curr. Opin. Immunol. 11:584-588, incorporated entirely by reference.Possible conjugate partners include but are not limited to cytokines,cytotoxic agents, toxins, radioisotopes, chemotherapeutic agent,anti-angiogenic agents, a tyrosine kinase inhibitors, and othertherapeutically active agents. In some embodiments, conjugate partnersmay be thought of more as payloads, that is to say that the goal of aconjugate is targeted delivery of the conjugate partner to a targetedcell, for example a cancer cell or immune cell, by the immunoglobulin.Thus, for example, the conjugation of a toxin to an immunoglobulintargets the delivery of said toxin to cells expressing the targetantigen. As will be appreciated by one skilled in the art, in realitythe concepts and definitions of fusion and conjugate are overlapping.The designation of a fusion or conjugate is not meant to constrain it toany particular embodiment disclosed herein. Rather, these terms are usedloosely to convey the broad concept that any immunoglobulin disclosedherein may be linked genetically, chemically, or otherwise, to one ormore polypeptides or molecules to provide some desirable property.

Suitable conjugates include, but are not limited to, labels as describedbelow, drugs and cytotoxic agents including, but not limited to,cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diptheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antibodies, or binding of a radionuclide to a chelatingagent that has been covalently attached to the antibody. Additionalembodiments utilize calicheamicin, auristatins, geldanamycin,maytansine, and duocarmycins and analogs; for the latter, see U.S.2003/0050331, incorporated entirely by reference.

In one embodiment, the immunoglobulins disclosed herein are fused orconjugated to a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. For example, as described inPenichet et al., 2001, J. Immunol. Methods 248:91-101, incorporatedentirely by reference, cytokines may be fused to antibody to provide anarray of desirable properties. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (I Ls) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; C5a; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture,and biologically active equivalents of the native sequence cytokines.

In an alternate embodiment, the immunoglobulins disclosed herein arefused, conjugated, or operably linked to a toxin, including but notlimited to small molecule toxins and enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof. For example, a variety of immunotoxins and immunotoxinmethods are described in Thrush et al., 1996, Ann. Rev. Immunol.14:49-71, incorporated entirely by reference. Small molecule toxinsinclude but are not limited to calicheamicin, maytansine (U.S. Pat. No.5,208,020, incorporated entirely by reference), trichothene, and CC1065.In one embodiment, an immunoglobulin disclosed herein may be conjugatedto one or more maytansine molecules (e.g. about 1 to about 10 maytansinemolecules per antibody molecule). Maytansine may, for example, beconverted to May-SS-Me which may be reduced to May-SH3 and reacted withmodified antibody (Chari et al., 1992, Cancer Research 52: 127-131,incorporated entirely by reference) to generate a maytansinoid-antibodyconjugate. Another conjugate of interest comprises an immunoglobulinconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. Structural analogues ofcalicheamicin that may be used include but are not limited to γ₁ ¹, α₂¹, α₃, N-acetyl-γ₁ ¹, PSAG, and ⊖¹ ₁, (Hinman et al., 1993, CancerResearch 53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928)(U.S. Pat. No. 5,714,586; U.S. Pat. No. 5,712,374; U.S. Pat. No.5,264,586; U.S. Pat. No. 5,773,001, all incorporated entirely byreference). Dolastatin 10 analogs such as auristatin E (AE) andmonomethylauristatin E (MMAE) may find use as conjugates for theimmunoglobulins disclosed herein (Doronina et al., 2003, Nat Biotechnol21(7):778-84; Francisco et al., 2003 Blood 102(4):1458-65, bothincorporated entirely by reference). Useful enyzmatically active toxinsinclude but are not limited to diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, for example, PCT WO 93/21232, incorporated entirely by reference.Embodiments further encompass a conjugate between an immunoglobulindisclosed herein and a compound with nucleolytic activity, for example aribonuclease or DNA endonuclease such as a deoxyribonuclease (DNase).

In an alternate embodiment, an immunoglobulin disclosed herein may befused, conjugated, or operably linked to a radioisotope to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugate antibodies. Examples include, but are notlimited to, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, andradioactive isotopes of Lu. See for example, reference.

In yet another embodiment, an immunoglobulin disclosed herein may beconjugated to a “receptor” (such streptavidin) for utilization in tumorpretargeting wherein the immunoglobulin-receptor conjugate isadministered to the patient, followed by removal of unbound conjugatefrom the circulation using a clearing agent and then administration of a“ligand” (e.g. avidin) which is conjugated to a cytotoxic agent (e.g. aradionucleotide). In an alternate embodiment, the immunoglobulin isconjugated or operably linked to an enzyme in order to employ AntibodyDependent Enzyme Mediated Prodrug Therapy (ADEPT). ADEPT may be used byconjugating or operably linking the immunoglobulin to aprodrug-activating enzyme that converts a prodrug (e.g. a peptidylchemotherapeutic agent, see PCT WO 81/01145, incorporated entirely byreference) to an active anti-cancer drug. See, for example, PCT WO88/07378 and U.S. Pat. No. 4,975,278, both incorporated entirely byreference. The enzyme component of the immunoconjugate useful for ADEPTincludes any enzyme capable of acting on a prodrug in such a way so asto convert it into its more active, cytotoxic form. Enzymes that areuseful in the method disclosed herein include but are not limited toalkaline phosphatase useful for converting phosphate-containing prodrugsinto free drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate-cleaving enzymes such as.beta.-galactosidase and neuramimidase useful for convertingglycosylated prodrugs into free drugs; beta-lactamase useful forconverting drugs derivatized with .alpha.-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes,” can be used to convert the prodrugsdisclosed herein into free active drugs (see, for example, Massey, 1987,Nature 328: 457-458, incorporated entirely by reference).immunoglobulin-abzyme conjugates can be prepared for delivery of theabzyme to a tumor cell population. A variety of additional conjugatesare contemplated for the immunoglobulins disclosed herein. A variety ofchemotherapeutic agents, anti-angiogenic agents, tyrosine kinaseinhibitors, and other therapeutic agents are described below, which mayfind use as immunoglobulin conjugates.

Conjugate partners may be linked to any region of an immunoglobulindisclosed herein, including at the N- or C-termini, or at some residuein-between the termini. A variety of linkers may find use inimmunoglobulins disclosed herein to covalently link conjugate partnersto an immunoglobulin. By “linker”, “linker sequence”, “spacer”,“tethering sequence” or grammatical equivalents thereof, herein is meanta molecule or group of molecules (such as a monomer or polymer) thatconnects two molecules and often serves to place the two molecules inone configuration. Linkers are known in the art; for example, homo- orhetero-bifunctional linkers as are well known (see, 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated entirely by reference). A number of strategies may be usedto covalently link molecules together. These include, but are notlimited to polypeptide linkages between N- and C-termini of proteins orprotein domains, linkage via disulfide bonds, and linkage via chemicalcross-linking reagents. In one aspect of this embodiment, the linker isa peptide bond, generated by recombinant techniques or peptidesynthesis. The linker peptide may predominantly include the followingamino acid residues: Gly, Ser, Ala, or Thr. The linker peptide shouldhave a length that is adequate to link two molecules in such a way thatthey assume the correct conformation relative to one another so thatthey retain the desired activity. Suitable lengths for this purposeinclude at least one and not more than 50 amino acid residues. In oneembodiment, the linker is from about 1 to 30 amino acids in length,e.g., a linker may be 1 to 20 amino acids in length. Useful linkersinclude glycine-serine polymers (including, for example, (GS)n, (GSGGS)n(Set forth as SEQ ID NO:1), (GGGGS)n (Set forth as SEQ ID NO:2) and(GGGS)n (Set forth as SEQ ID NO:3), where n is an integer of at leastone), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers, as will be appreciated by those in the art.Alternatively, a variety of nonproteinaceous polymers, including but notlimited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may find use as linkers.

Production of Immunoglobulins

Also disclosed herein are methods for producing and experimentallytesting immunoglobulins. The disclosed methods are not meant toconstrain embodiments to any particular application or theory ofoperation. Rather, the provided methods are meant to illustrategenerally that one or more immunoglobulins may be produced andexperimentally tested to obtain immunoglobulins. General methods forantibody molecular biology, expression, purification, and screening aredescribed in Antibody Engineering, edited by Duebel & Kontermann,Springer-Verlag, Heidelberg, 2001; and Hayhurst & Georgiou, 2001, CurrOpin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng2:339-76; Antibodies: A Laboratory Manual by Harlow & Lane, New York:Cold Spring Harbor Laboratory Press, 1988, all incorporated entirely byreference.

In one embodiment disclosed herein, nucleic acids are created thatencode the immunoglobulins, and that may then be cloned into host cells,expressed and assayed, if desired. Thus, nucleic acids, and particularlyDNA, may be made that encode each protein sequence. These practices arecarried out using well-known procedures. For example, a variety ofmethods that may find use in generating immunoglobulins disclosed hereinare described in Molecular Cloning—A Laboratory Manual, 3^(1d) Ed.(Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), andCurrent Protocols in Molecular Biology (John Wiley & Sons), bothincorporated entirely by reference. As will be appreciated by thoseskilled in the art, the generation of exact sequences for a librarycomprising a large number of sequences is potentially expensive and timeconsuming. By “library” herein is meant a set of variants in any form,including but not limited to a list of nucleic acid or amino acidsequences, a list of nucleic acid or amino acid substitutions atvariable positions, a physical library comprising nucleic acids thatencode the library sequences, or a physical library comprising thevariant proteins, either in purified or unpurified form. Accordingly,there are a variety of techniques that may be used to efficientlygenerate libraries disclosed herein. Such methods that may find use forgenerating immunoglobulins disclosed herein are described or referencedin U.S. Pat. No. 6,403,312; US Pub. No. 2002/0048772; U.S. Pat. No.7,315,786; US Pub. No. 2003/0130827; PCT WO 01/40091; and PCT WO02/25588, all incorporated entirely by reference. Such methods includebut are not limited to gene assembly methods, PCR-based method andmethods which use variations of PCR, ligase chain reaction-basedmethods, pooled oligo methods such as those used in synthetic shuffling,error-prone amplification methods and methods which use oligos withrandom mutations, classical site-directed mutagenesis methods, cassettemutagenesis, and other amplification and gene synthesis methods. As isknown in the art, there are a variety of commercially available kits andmethods for gene assembly, mutagenesis, vector subcloning, and the like,and such commercial products find use in for generating nucleic acidsthat encode immunoglobulins.

The immunoglobulins disclosed herein may be produced by culturing a hostcell transformed with nucleic acid, e.g., an expression vector,containing nucleic acid encoding the immunoglobulins, under theappropriate conditions to induce or cause expression of the protein. Theconditions appropriate for expression will vary with the choice of theexpression vector and the host cell, and will be easily ascertained byone skilled in the art through routine experimentation. A wide varietyof appropriate host cells may be used, including but not limited tomammalian cells, bacteria, insect cells, and yeast. For example, avariety of cell lines that may find use in generating immunoglobulinsdisclosed herein are described in the ATCC® cell line catalog, availablefrom the American Type Culture Collection.

In one embodiment, the immunoglobulins are expressed in mammalianexpression systems, including systems in which the expression constructsare introduced into the mammalian cells using virus such as retrovirusor adenovirus. Any mammalian cells may be used, e.g., human, mouse, rat,hamster, and primate cells. Suitable cells also include known researchcells, including but not limited to Jurkat T cells, NIH3T3, CHO, BHK,COS, HEK293, PER C.6, HeLa, Sp2/0, NS0 cells, and variants thereof. Inan alternate embodiment, library proteins are expressed in bacterialcells. Bacterial expression systems are well known in the art, andinclude Escherichia coli (E. coli), Bacillus subtilis, Streptococcuscremoris, and Streptococcus lividans. In alternate embodiments,immunoglobulins are produced in insect cells (e.g. Sf21/Sf9,Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g. S. cerevisiae, Pichia,etc). In an alternate embodiment, immunoglobulins are expressed in vitrousing cell free translation systems. In vitro translation systemsderived from both prokaryotic (e.g. E. coli) and eukaryotic (e.g. wheatgerm, rabbit reticulocytes) cells are available and may be chosen basedon the expression levels and functional properties of the protein ofinterest. For example, as appreciated by those skilled in the art, invitro translation is required for some display technologies, for exampleribosome display. In addition, the immunoglobulins may be produced bychemical synthesis methods. Also transgenic expression systems bothanimal (e.g. cow, sheep or goat milk, embryonated hen's eggs, wholeinsect larvae, etc.) and plant (e.g. corn, tobacco, duckweed, etc.)

The nucleic acids that encode the immunoglobulins disclosed herein maybe incorporated into an expression vector in order to express theprotein. A variety of expression vectors may be utilized for proteinexpression. Expression vectors may comprise self-replicatingextra-chromosomal vectors or vectors which integrate into a host genome.Expression vectors are constructed to be compatible with the host celltype. Thus expression vectors which find use in generatingimmunoglobulins disclosed herein include but are not limited to thosewhich enable protein expression in mammalian cells, bacteria, insectcells, yeast, and in in vitro systems. As is known in the art, a varietyof expression vectors are available, commercially or otherwise, that mayfind use for expressing immunoglobulins disclosed herein.

Expression vectors typically comprise a protein operably linked withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. By “operably linked” herein ismeant that the nucleic acid is placed into a functional relationshipwith another nucleic acid sequence. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the immunoglobulin, and aretypically appropriate to the host cell used to express the protein. Ingeneral, the transcriptional and translational regulatory sequences mayinclude promoter sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and stop sequences, andenhancer or activator sequences. As is also known in the art, expressionvectors typically contain a selection gene or marker to allow theselection of transformed host cells containing the expression vector.Selection genes are well known in the art and will vary with the hostcell used.

Immunoglobulins may be operably linked to a fusion partner to enabletargeting of the expressed protein, purification, screening, display,and the like. Fusion partners may be linked to the immunoglobulinsequence via a linker sequences. The linker sequence will generallycomprise a small number of amino acids, typically less than ten,although longer linkers may also be used. Typically, linker sequencesare selected to be flexible and resistant to degradation. As will beappreciated by those skilled in the art, any of a wide variety ofsequences may be used as linkers. For example, a common linker sequencecomprises the amino acid sequence GGGGS. A fusion partner may be atargeting or signal sequence that directs immunoglobulin and anyassociated fusion partners to a desired cellular location or to theextracellular media. As is known in the art, certain signaling sequencesmay target a protein to be either secreted into the growth media, orinto the periplasmic space, located between the inner and outer membraneof the cell. A fusion partner may also be a sequence that encodes apeptide or protein that enables purification and/or screening. Suchfusion partners include but are not limited to polyhistidine tags(His-tags) (for example H₆ and H₁₀ or other tags for use withImmobilized Metal Affinity Chromatography (IMAC) systems (e.g. Ni⁺²affinity columns)), GST fusions, MBP fusions, Strep-tag, the BSPbiotinylation target sequence of the bacterial enzyme BirA, and epitopetags which are targeted by antibodies (for example c-myc tags,flag-tags, and the like). As will be appreciated by those skilled in theart, such tags may be useful for purification, for screening, or both.For example, an immunoglobulin may be purified using a His-tag byimmobilizing it to a Ni⁺² affinity column, and then after purificationthe same His-tag may be used to immobilize the antibody to a Ni⁺² coatedplate to perform an ELISA or other binding assay (as described below). Afusion partner may enable the use of a selection method to screenimmunoglobulins (see below). Fusion partners that enable a variety ofselection methods are well-known in the art. For example, by fusing themembers of an immunoglobulin library to the gene III protein, phagedisplay can be employed (Kay et al., Phage display of peptides andproteins: a laboratory manual, Academic Press, San Diego, Calif., 1996;Lowman et al., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science228:1315-1317, incorporated entirely by reference). Fusion partners mayenable immunoglobulins to be labeled. Alternatively, a fusion partnermay bind to a specific sequence on the expression vector, enabling thefusion partner and associated immunoglobulin to be linked covalently ornoncovalently with the nucleic acid that encodes them. The methods ofintroducing exogenous nucleic acid into host cells are well known in theart, and will vary with the host cell used. Techniques include but arenot limited to dextran-mediated transfection, calcium phosphateprecipitation, calcium chloride treatment, polybrene mediatedtransfection, protoplast fusion, electroporation, viral or phageinfection, encapsulation of the polynucleotide(s) in liposomes, anddirect microinjection of the DNA into nuclei. In the case of mammaliancells, transfection may be either transient or stable.

In one embodiment, immunoglobulins are purified or isolated afterexpression. Proteins may be isolated or purified in a variety of waysknown to those skilled in the art. Standard purification methods includechromatographic techniques, including ion exchange, hydrophobicinteraction, affinity, sizing or gel filtration, and reversed-phase,carried out at atmospheric pressure or at high pressure using systemssuch as FPLC and HPLC. Purification methods also includeelectrophoretic, immunological, precipitation, dialysis, andchromatofocusing techniques. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.As is well known in the art, a variety of natural proteins bind Fc andantibodies, and these proteins can find use for purification ofimmunoglobulins disclosed herein. For example, the bacterial proteins Aand G bind to the Fc region. Likewise, the bacterial protein L binds tothe Fab region of some antibodies, as of course does the antibody'starget antigen. Purification can often be enabled by a particular fusionpartner. For example, immunoglobulins may be purified using glutathioneresin if a GST fusion is employed, Ni⁺² affinity chromatography if aHis-tag is employed, or immobilized anti-flag antibody if a flag-tag isused. For general guidance in suitable purification techniques, see,e.g. incorporated entirely by reference Protein Purification: Principlesand Practice, 3^(rd) Ed., Scopes, Springer-Verlag, NY, 1994,incorporated entirely by reference. The degree of purification necessarywill vary depending on the screen or use of the immunoglobulins. In someinstances no purification is necessary. For example in one embodiment,if the immunoglobulins are secreted, screening may take place directlyfrom the media. As is well known in the art, some methods of selectiondo not involve purification of proteins. Thus, for example, if a libraryof immunoglobulins is made into a phage display library, proteinpurification may not be performed.

In Vitro Experimentation

Immunoglobulins may be screened using a variety of methods, includingbut not limited to those that use in vitro assays, in vivo andcell-based assays, and selection technologies. Automation andhigh-throughput screening technologies may be utilized in the screeningprocedures. Screening may employ the use of a fusion partner or label.The use of fusion partners has been discussed above. By “labeled” hereinis meant that the immunoglobulins disclosed herein have one or moreelements, isotopes, or chemical compounds attached to enable thedetection in a screen. In general, labels fall into three classes: a)immune labels, which may be an epitope incorporated as a fusion partnerthat is recognized by an antibody, b) isotopic labels, which may beradioactive or heavy isotopes, and c) small molecule labels, which mayinclude fluorescent and colorimetric dyes, or molecules such as biotinthat enable other labeling methods. Labels may be incorporated into thecompound at any position and may be incorporated in vitro or in vivoduring protein expression.

In one embodiment, the functional and/or biophysical properties ofimmunoglobulins are screened in an in vitro assay. In vitro assays mayallow a broad dynamic range for screening properties of interest.Properties of immunoglobulins that may be screened include but are notlimited to stability, solubility, and affinity for Fc ligands, forexample FcγRs. Multiple properties may be screened simultaneously orindividually. Proteins may be purified or unpurified, depending on therequirements of the assay. In one embodiment, the screen is aqualitative or quantitative binding assay for binding of immunoglobulinsto a protein or nonprotein molecule that is known or thought to bind theimmunoglobulin. In one embodiment, the screen is a binding assay formeasuring binding to the target antigen. In an alternate embodiment, thescreen is an assay for binding of immunoglobulins to an Fc ligand,including but are not limited to the family of FcγRs, the neonatalreceptor FcRn, the complement protein C1q, and the bacterial proteins Aand G. Said Fc ligands may be from any organism. In one embodiment, Fcligands are from humans, mice, rats, rabbits, and/or monkeys. Bindingassays can be carried out using a variety of methods known in the art,including but not limited to FRET (Fluorescence Resonance EnergyTransfer) and BRET (Bioluminescence Resonance Energy Transfer)-basedassays, AlphaScreen™ (Amplified Luminescent Proximity HomogeneousAssay), Scintillation Proximity Assay, ELISA (Enzyme-LinkedImmunosorbent Assay), SPR (Surface Plasmon Resonance, also known asBIACORE®), isothermal titration calorimetry, differential scanningcalorimetry, gel electrophoresis, and chromatography including gelfiltration. These and other methods may take advantage of some fusionpartner or label of the immunoglobulin. Assays may employ a variety ofdetection methods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels.

The biophysical properties of immunoglobulins, for example stability andsolubility, may be screened using a variety of methods known in the art.Protein stability may be determined by measuring the thermodynamicequilibrium between folded and unfolded states. For example,immunoglobulins disclosed herein may be unfolded using chemicaldenaturant, heat, or pH, and this transition may be monitored usingmethods including but not limited to circular dichroism spectroscopy,fluorescence spectroscopy, absorbance spectroscopy, NMR spectroscopy,calorimetry, and proteolysis. As will be appreciated by those skilled inthe art, the kinetic parameters of the folding and unfolding transitionsmay also be monitored using these and other techniques. The solubilityand overall structural integrity of an immunoglobulin may bequantitatively or qualitatively determined using a wide range of methodsthat are known in the art. Methods which may find use for characterizingthe biophysical properties of immunoglobulins disclosed herein includegel electrophoresis, isoelectric focusing, capillary electrophoresis,chromatography such as size exclusion chromatography, ion-exchangechromatography, and reversed-phase high performance liquidchromatography, peptide mapping, oligosaccharide mapping, massspectrometry, ultraviolet absorbance spectroscopy, fluorescencespectroscopy, circular dichroism spectroscopy, isothermal titrationcalorimetry, differential scanning calorimetry, analyticalultra-centrifugation, dynamic light scattering, proteolysis, andcross-linking, turbidity measurement, filter retardation assays,immunological assays, fluorescent dye binding assays, protein-stainingassays, microscopy, and detection of aggregates via ELISA or otherbinding assay. Structural analysis employing X-ray crystallographictechniques and NMR spectroscopy may also find use. In one embodiment,stability and/or solubility may be measured by determining the amount ofprotein solution after some defined period of time. In this assay, theprotein may or may not be exposed to some extreme condition, for exampleelevated temperature, low pH, or the presence of denaturant. Becausefunction typically requires a stable, soluble, and/orwell-folded/structured protein, the aforementioned functional andbinding assays also provide ways to perform such a measurement. Forexample, a solution comprising an immunoglobulin could be assayed forits ability to bind target antigen, then exposed to elevated temperaturefor one or more defined periods of time, then assayed for antigenbinding again. Because unfolded and aggregated protein is not expectedto be capable of binding antigen, the amount of activity remainingprovides a measure of the immunoglobulin's stability and solubility.

In one embodiment, the library is screened using one or more cell-basedor in vitro assays. For such assays, immunoglobulins, purified orunpurified, are typically added exogenously such that cells are exposedto individual variants or groups of variants belonging to a library.These assays are typically, but not always, based on the biology of theability of the immunoglobulin to bind to the target antigen and mediatesome biochemical event, for example effector functions like cellularlysis, phagocytosis, ligand/receptor binding inhibition, inhibition ofgrowth and/or proliferation, apoptosisand the like. Such assays ofteninvolve monitoring the response of cells to immunoglobulin, for examplecell survival, cell death, cellular phagocytosis, cell lysis, change incellular morphology, or transcriptional activation such as cellularexpression of a natural gene or reporter gene. For example, such assaysmay measure the ability of immunoglobulins to elicit ADCC, ADCP, or CDC.For some assays additional cells or components, that is in addition tothe target cells, may need to be added, for example serum complement, oreffector cells such as peripheral blood monocytes (PBMCs), NK cells,macrophages, and the like. Such additional cells may be from anyorganism, e.g., humans, mice, rat, rabbit, and monkey. Crosslinked ormonomeric antibodies may cause apoptosis of certain cell linesexpressing the antibody's target antigen, or they may mediate attack ontarget cells by immune cells which have been added to the assay. Methodsfor monitoring cell death or viability are known in the art, and includethe use of dyes, fluorophores, immunochemical, cytochemical, andradioactive reagents. For example, caspase assays orannexin-flourconjugates may enable apoptosis to be measured, and uptakeor release of radioactive substrates (e.g. Chromium-51 release assays)or the metabolic reduction of fluorescent dyes such as alamar blue mayenable cell growth, proliferation, or activation to be monitored. In oneembodiment, the DELFIA® EuTDA-based cytotoxicity assay (Perkin Elmer,MA) is used. Alternatively, dead or damaged target cells may bemonitored by measuring the release of one or more natural intracellularproteins, for example lactate dehydrogenase. Transcriptional activationmay also serve as a method for assaying function in cell-based assays.In this case, response may be monitored by assaying for natural genes orproteins which may be upregulated or down-regulated, for example therelease of certain interleukins may be measured, or alternativelyreadout may be via a luciferase or GFP-reporter construct. Cell-basedassays may also involve the measure of morphological changes of cells asa response to the presence of an immunoglobulin. Cell types for suchassays may be prokaryotic or eukaryotic, and a variety of cell linesthat are known in the art may be employed. Alternatively, cell-basedscreens are performed using cells that have been transformed ortransfected with nucleic acids encoding the immunoglobulins.

In vitro assays include but are not limited to binding assays, ADCC,CDC, cytotoxicity, proliferation, peroxide/ozone release, chemotaxis ofeffector cells, inhibition of such assays by reduced effector functionantibodies; ranges of activities such as >100× improvement or >100×reduction, blends of receptor activation and the assay outcomes that areexpected from such receptor profiles.

In Vivo Experimentation

The biological properties of the immunoglobulins disclosed herein may becharacterized in cell, tissue, and whole organism experiments. As isknown in the art, drugs are often tested in animals, including but notlimited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in orderto measure a drug's efficacy for treatment against a disease or diseasemodel, or to measure a drug's pharmacokinetics, toxicity, and otherproperties. Said animals may be referred to as disease models. Withrespect to the immunoglobulins disclosed herein, a particular challengearises when using animal models to evaluate the potential for in-humanefficacy of candidate polypeptides—this is due, at least in part, to thefact that immunoglobulins that have a specific effect on the affinityfor a human Fc receptor may not have a similar affinity effect with theorthologous animal receptor. These problems can be further exacerbatedby the inevitable ambiguities associated with correct assignment of trueorthologues (Mechetina et al., Immunogenetics, 2002 54:463-468,incorporated entirely by reference), and the fact that some orthologuessimply do not exist in the animal (e.g. humans possess an FcγRIIawhereas mice do not). Therapeutics are often tested in mice, includingbut not limited to mouse strains NZB, NOD, BXSB, MRL/Ipr, K/BxN andtransgenics (including knockins and knockouts). Such mice can developvarious autoimmune conditions that resemble human organ specific,systemic autoimmune or inflammatory disease pathologies such as systemiclupus erythematosus (SLE) and rheumatoid arthritis (RA). For example, animmunoglobulin disclosed herein intended for autoimmune diseases may betested in such mouse models by treating the mice to determine theability of the immunoglobulin to reduce or inhibit the development ofthe disease pathology. Because of the incompatibility between the mouseand human Fcγ receptor system, an alternative approach is to use amurine SCID model in which immune deficient mice are engrafted withhuman PBLs or PBMCs (huPBL-SCID, huPBMC-SCID) providing asemi-functional human immune system with human effector cells and Fcreceptors. In such a model, an antigen challenge (such as tetanustoxoid) activates the B cells to differentiate into plasma cells andsecrete immunoglobulins, thus reconstituting antigen specific humoralimmunity. Therefore, a dual targeting immunoglobulin disclosed hereinthat specifically binds to an antigen (such as CD19 or CD79a/b) andFcγRIIb on B cells may be tested to examine the ability to specificallyinhibit B cell differentiation. Such experimentation may providemeaningful data for determination of the potential of saidimmunoglobulin to be used as a therapeutic. Other organisms, e.g.,mammals, may also be used for testing. For example, because of theirgenetic similarity to humans, monkeys can be suitable therapeuticmodels, and thus may be used to test the efficacy, toxicity,pharmacokinetics, or other property of the immunoglobulins disclosedherein. Tests of the immunoglobulins disclosed herein in humans areultimately required for approval as drugs, and thus of course theseexperiments are contemplated. Thus the immunoglobulins disclosed hereinmay be tested in humans to determine their therapeutic efficacy,toxicity, pharmacokinetics, and/or other clinical properties.

The immunoglobulins disclosed herein may confer superior performance onFc-containing therapeutics in animal models or in humans. The receptorbinding profiles of such immunoglobulins, as described in thisspecification, may, for example, be selected to increase the potency ofcytotoxic drugs or to target specific effector functions or effectorcells to improve the selectivity of the drug's action. Further, receptorbinding profiles can be selected that may reduce some or all effectorfunctions thereby reducing the side-effects or toxicity of suchFc-containing drug. For example, an immunoglobulin with reduced bindingto FcγRIIIa, FcγRI, and FcγRIIa can be selected to eliminate mostcell-mediated effector function, or an immunoglobulin with reducedbinding to C1q may be selected to limit complement-mediated effectorfunctions. In some contexts, such effector functions are known to havepotential toxic effects. Therefore eliminating them may increase thesafety of the Fc-bearing drug and such improved safety may becharacterized in animal models. In some contexts, such effectorfunctions are known to mediate the desirable therapeutic activity.Therefore enhancing them may increase the activity or potency of theFc-bearing drug and such improved activity or potency may becharacterized in animal models.

In some embodiments, immunoglobulins disclosed herein may be assessedfor efficacy in clinically relevant animal models of various humandiseases. In many cases, relevant models include various transgenicanimals for specific antigens and receptors.

Relevant transgenic models such as those that express human Fc receptors(e.g., CD32b) could be used to evaluate and test immunoglobulins andFc-fusions in their efficacy. The evaluation of immunoglobulins by theintroduction of human genes that directly or indirectly mediate effectorfunction in mice or other rodents may enable physiological studies ofefficacy in autoimmune disorders and RA. Human Fc receptors such asFcγRIIb may possess polymorphisms such as that in gene promoter (−343from G to C) or transmembrane domain of the receptor 187 I or T whichwould further enable the introduction of specific and combinations ofhuman polymorphisms into rodents. The various studies involvingpolymorphism-specific FcRs is not limited to this section, howeverencompasses all discussions and applications of FcRs in general asspecified in throughout this application. Immunoglobulins disclosedherein may confer superior activity on Fc-containing drugs in suchtransgenic models, in particular variants with binding profilesoptimized for human FcγRIIb mediated activity may show superior activityin transgenic CD32b mice. Similar improvements in efficacy in micetransgenic for the other human Fc receptors, e.g. FcγRIIa, FcγRI, etc.,may be observed for immunoglobulins with binding profiles optimized forthe respective receptors. Mice transgenic for multiple human receptorswould show improved activity for immunoglobulins with binding profilesoptimized for the corresponding multiple receptors.

Because of the difficulties and ambiguities associated with using animalmodels to characterize the potential efficacy of candidate therapeuticantibodies in a human patient, some variant polypeptides disclosedherein may find utility as proxies for assessing potential in-humanefficacy. Such proxy molecules may mimic—in the animal system—the FcRand/or complement biology of a corresponding candidate humanimmunoglobulin. This mimicry is most likely to be manifested by relativeassociation affinities between specific immunoglobulins and animal vs.human receptors. For example, if one were using a mouse model to assessthe potential in-human efficacy of an Fc variant that has reducedaffinity for the inhibitory human FcγRIIb, an appropriate proxy variantwould have reduced affinity for mouse FcγRII. It should also be notedthat the proxy Fc variants could be created in the context of a human Fcvariant, an animal Fc variant, or both.

In one embodiment, the testing of immunoglobulins may include study ofefficacy in primates (e.g. cynomolgus monkey model) to facilitate theevaluation of reduction and/or depletion of specific target cellsharboring the target antigen. Additional primate models include but arenot limited to use of the rhesus monkey to assess Fc polypeptides intherapeutic studies of autoimmune, transplantation and cancer.

Toxicity studies are performed to determine antibody or Fc-fusionrelated-effects that cannot be evaluated in standard pharmacologyprofiles, or occur only after repeated administration of the agent. Mosttoxicity tests are performed in two species—a rodent and a non-rodent—toensure that any unexpected adverse effects are not overlooked before newtherapeutic entities are introduced into man. In general, these modelsmay measure a variety of toxicities including genotoxicity, chronictoxicity, immunogenicity, reproductive/developmental toxicity, andcarcinogenicity. Included within the aforementioned parameters arestandard measurement of food consumption, bodyweight, antibodyformation, clinical chemistry, and macro- and microscopic examination ofstandard organs/tissues (e.g. cardiotoxicity). Additional parameters ofmeasurement are injection site trauma and the measurement ofneutralizing antibodies, if any. Traditionally, monoclonal antibodytherapeutics, naked or conjugated, are evaluated for cross-reactivitywith normal tissues, immunogenicity/antibody production, conjugate orlinker toxicity and “bystander” toxicity of radiolabelled species.Nonetheless, such studies may have to be individualized to addressspecific concerns and following the guidance set by ICH S6 (Safetystudies for biotechnological products, also noted above). As such, thegeneral principles are that the products are sufficiently wellcharacterized, impurities/contaminants have been removed, that the testmaterial is comparable throughout development, that GLP compliance ismaintained.

The pharmacokinetics (PK) of the immunoglobulins disclosed herein may bestudied in a variety of animal systems, with the most relevant beingnon-human primates such as the cynomolgus and rhesus monkeys. Single orrepeated IV/SC administrations over a dose range of 6000-fold (0.05-300mg/kg) can be evaluated for half-life (days to weeks) using plasmaconcentration and clearance. Volume of distribution at a steady stateand level of systemic absorbance can also be measured. Examples of suchparameters of measurement generally include maximum observed plasmaconcentration (Cmax), the time to reach Cmax (Tmax), the area under theplasma concentration-time curve from time 0 to infinity [AUC(0-inf] andapparent elimination half-life (T1/2). Additional measured parameterscould include compartmental analysis of concentration-time data obtainedfollowing IV administration and bioavailability. Examples ofpharmacological/toxicological studies using cynomolgus monkeys have beenestablished for Rituxan and Zevalin in which monoclonal antibodies toCD20 are cross-reactive. Biodistribution, dosimetry (for radiolabelledantibodies), and PK studies can also be done in rodent models. Suchstudies would evaluate tolerance at all doses administered, toxicity tolocal tissues, preferential localization to rodent xenograft animalmodels, and reduction and/or depletion of target cells (e.g. CD20positive cells).

The immunoglobulins disclosed herein may confer superiorpharmacokinetics on Fc-containing therapeutics in animal systems or inhumans. For example, increased binding to FcRn may increase thehalf-life and exposure of the Fc-containing drug. Alternatively,decreased binding to FcRn may decrease the half-life and exposure of theFc-containing drug in cases where reduced exposure is favorable such aswhen such drug has side-effects.

It is known in the art that the array of Fc receptors is differentiallyexpressed on various immune cell types, as well as in different tissues.Differential tissue distribution of Fc receptors may ultimately have animpact on the pharmacodynamic (PD) and pharmacokinetic (PK) propertiesof immunoglobulins disclosed herein. Because immunoglobulins of thepresentation have varying affinities for the array of Fc receptors,further screening of the polypeptides for PD and/or PK properties may beextremely useful for defining the optimal balance of PD, PK, andtherapeutic efficacy conferred by each candidate polypeptide.

Pharmacodynamic studies may include, but are not limited to, targetingspecific cells or blocking signaling mechanisms, measuring inhibition ofantigen-specific antibodies etc. The immunoglobulins disclosed hereinmay target particular effector cell populations and thereby directFc-containing drugs to induce certain activities to improve potency orto increase penetration into a particularly favorable physiologicalcompartment. For example, neutrophil activity and localization can betargeted by an immunoglobulin that targets FcγRIIIb. Suchpharmacodynamic effects may be demonstrated in animal models or inhumans.

Clinical Use

The immunoglobulins disclosed herein may find use in a wide range ofproducts. In one embodiment an immunoglobulin disclosed herein is atherapeutic, a diagnostic, or a research reagent. The immunoglobulinsmay find use in a composition that is monoclonal or polyclonal. Theimmunoglobulins disclosed herein may be used for therapeutic purposes.As will be appreciated by those in the art, the immunoglobulinsdisclosed herein may be used for any therapeutic purpose thatantibodies, and the like may be used for. The immunoglobulins may beadministered to a patient to treat IgG4-related disease.

A “patient” for the purposes disclosed herein includes both humans andother animals, e.g., other mammals. Thus the immunoglobulins disclosedherein have both human therapy and veterinary applications. The term“treatment” or “treating” as disclosed herein is meant to includetherapeutic treatment, as well as prophylactic, or suppressive measuresfor a disease or disorder. Thus, for example, successful administrationof an immunoglobulin prior to onset of the disease results in treatmentof the disease. As another example, successful administration of anoptimized immunoglobulin after clinical manifestation of the disease tocombat the symptoms of the disease comprises treatment of the disease.“Treatment” and “treating” also encompasses administration of anoptimized immunoglobulin after the appearance of the disease in order toeradicate the disease. Successful administration of an agent after onsetand after clinical symptoms have developed, with possible abatement ofclinical symptoms and perhaps amelioration of the disease, comprisestreatment of the disease. Those “in need of treatment” include mammalsalready having the disease or disorder, as well as those prone to havingthe disease or disorder, including those in which the disease ordisorder is to be prevented.

Immunoglobulins disclosed herein can be used to treat IgG4-relateddisease (also referred to as “IgG4-RD”). IgG4-RD is also known as orreferred to as IgG4-related systemic disease (IgG4-RSD), IgG4-relatedsclerosing disease, IgG4-related systemic sclerosing disease,IgG4-related autoimmune disease, IgG4-associated multifocal systemicfibrosis, IgG4-associated disease, IgG4 syndrome, hyper-IgG4 disease,systemic IgG4-related plasmacytic syndrome, IgG4-positive multiorganlymphoproliferative syndrome (IgG4-related mutli-organlympoproliferatice syndrome (IgG4-MOLPS), systemic IgG4-relatedsclerosing syndrome, multifocal fibrosclerosis, and multifocalidiopathic fibrosclerosis.

IgG4-RD is a systemic inflammatory condition that can affect any organsystem. Immunoglobulins disclosed herein can also be used to treatmanifestations of IgG4-RD or IgG4-RD related diseases. Non-limitingexamples of manifestations related to IgG4-RD and/or IgG4-RD relateddiseases include: IgG4-related sialadenitis (chronic sclerosingsialadenitis, Küttner's tumour, Mikulicz's disease), IgG4-relateddacryoadenitis (Mikulicz's disease), IgG4-related ophthalmic disease(idiopathic orbital inflammatory disease, orbital pseudotumor), chronicsinusitis, eosinophilic angiocentric fibrosis, IgG4-related hypophysitis(IgG4-related panhypophysitis, IgG4-related adenohypophysitis,gG4-related infundibuloneurohypophysitis, autoimmune hypophysitis),IgG4-related pachymeningitis, IgG4-related leptomeningitis (idiopathichypertrophic pachymeningitis), IgG4-related pancreatitis (Type 1autoimmune pancreatitis, IgG4-related AIP, lymphoplasmacytic sclerosingpancreatitis, chronic pancreatitis with diffuse irregular narrowing ofthe main pancreatic duct), IgG4-related lung disease (Pulmonaryinflammatory pseudotumour), IgG4-related pleuritis, IgG4-relatedhepatopathy, IgG4-related sclerosing cholangitis, IgG4-relatedcholecystitis, IgG4-related aortitis (inflammatory aortic aneurysm),IgG4-related periaortitis (chronic periaortitis), IgG4-relatedperiarteritis, IgG4-related pericarditis, IgG4-related mediastinitis(fibrosing mediastinitis), IgG4-related retroperitoneal fibrosis(retroperitoneal fibrosis, Albarran-Ormond syndrome, Ormond's disease(tetroperitoneal fibrosis)), perirenal fasciitis, Gerota'sfasciitis/syndrome, periureteritis fibrosa, sclerosing lipogranuloma,sclerosing retroperitoneal granuloma, non-specific retroperitonealinflammation, sclerosing retroperitonitis, retroperitoneal vasculitiswith perivascular fibrosis), IgG4-related mesenteritis (subtypes are:mesenteric panniculitis, mesenteric lipodystrophy and retractilemesenteritis) (sclerosing mesenteritis, systemic nodular panniculitis,liposclerosis mesenteritis, mesenteric Weber-Christian disease,mesenteric lipogranuloma, xanthogranulomatous mesenteritis),IgG4-related mastitis (sclerosing mastitis), IgG4-related kidney disease(IgG4-RKD), IgG4-related tubulointerstitial nephritis (IgG4-TIN),IgG4-related membranous glomerulonephritis (idiopathictubulointerstitial nephritis), IgG4-related prostatitis, IgG4-relatedperivasal fibrosis (chronic orchialgia), IgG4-related paratesticularpseudotumor, IgG4-related epididymo-orchitis (paratesticular fibrouspseudotumor, inflammatory pseudotumor of the spermatic cord,pseudosarcomatous myofibroblastic proliferations of the spermatic cord,proliferative funiculitis, chronic proliferative periorchitis,fibromatous periorchitis, nodular periorchitis, reactive periorchitis,fibrous mesothelioma), IgG4-related lymphadenopathy, IgG4-related skindisease (angiolymphoid hyperplasia with eosinophilia, cutaneouspseudolymphoma), IgG4-related perineural disease, and IgG4-relatedthyroid disease (Reidel's thyroiditis), eosinophilic angiocentricfibrosis (affecting the orbits and upper respiratory tract),inflammatory pseudotumour, and multifocal fibrosclerosis.

More specifically, in some embodiments, immunoglobulins disclosed hereincan be used to treat autoimmune pancreatitis (lymphoplasmacyticscleorising pancreatitis), eosinophilic angiocentric fibrosis (affectingthe orbits and upper respiratory tract), fibrosing mediastinitis,idiopathic hypertrophic pachymeningitis, idiopathic tubulointerstitialnephritis, inflammatory pseudotumour, Küttner's tumour, Mikulicz'sdisease, multifocal fibrosclerosis, periaortitis, periarteritis,inflammatory aortic aneurysm, Ormond's disease (tetroperitonealfibrosis), Riedel's thyroiditis, and sclerosing mesenteritis.

In further embodiments, the immunoglobulins disclosed herein can be usedto treat autoimmune pancreatitis (lymphoplasmacytic scleorisingpancreatitis). In other embodiments, the immunoglobulins disclosedherein can be used to treat eosinophilic angiocentric fibrosis(affecting the orbits and upper respiratory tract). In otherembodiments, the immunoglobulins disclosed herein can be used to treatfibrosing mediastinitis. In other embodiments, the immunoglobulinsdisclosed herein can be used to treat idiopathic hypertrophicpachymeningitis. In other embodiments, the immunoglobulins disclosedherein can be used to treat idiopathic tubulointerstitial nephritis. Inother embodiments, the immunoglobulins disclosed herein can be used totreat inflammatory pseudotumour. In other embodiments, theimmunoglobulins disclosed herein can be used to treat Küttner's tumour.In other embodiments, the immunoglobulins disclosed herein can be usedto treat Mikulicz's disease. In other embodiments, the immunoglobulinsdisclosed herein can be used to treat. In other embodiments, theimmunoglobulins disclosed herein can be used to treat multifocalfibrosclerosis. In other embodiments, the immunoglobulins disclosedherein can be used to treat periaortitis. In other embodiments, theimmunoglobulins disclosed herein can be used to treat periarteritis. Inother embodiments, the immunoglobulins disclosed herein can be used totreat inflammatory aortic aneurysm. In other embodiments, theimmunoglobulins disclosed herein can be used to treat Ormond's disease(tetroperitoneal fibrosis). In other embodiments, the immunoglobulinsdisclosed herein can be used to treat Riedel's thyroiditis. In otherembodiments, the immunoglobulins disclosed herein can be used to treatsclerosing mesenteritis.

Without wishing to be bound by theory, an immunoglobulin of thedisclosure for use in the treatment of an IgG4-RD coengages FcγRIIb andCD19 expressed on B cells. The coengagement of FcγRIIb and CD19 inhibitsactivation of the B cell and prevents or limits the production ofantibodies. Specifically, the coengagement of FcγRIIb and CD19 inhibitsor reduces the production of antigen specific antibodies.

As stated above, immunoglobulins disclosed herein can be used to treatIgG4-RD and manifestations and/or diseases and conditions associatedwith IgG4-RD (such as, but not limited to, those listed previously). Insome aspects, treating can treat symptoms associated with IgG4-RD. Insome aspects, treating can prevent IgG4-RD. In some aspects, treatingcan be prophylactic treatment for IgG4-RD.

The immunoglobulins disclosed herein can be used to inhibit B cells forthe treatment of IgG4-RD. An antibody used to treat IgG4-RD can be animmunoglobulin comprising a domain that binds FcγRIIb and a domain thatbinds CD19. More specifically, an immunoglobulin used to treat IgG4-RDcan be an antibody comprising a Fc domain that binds FcγRIIb and a Fvdomain that binds CD19.

In some embodiments, the Fc domain that binds FcγRIIb comprises at leastone modification selected from 234W, 235I, 235Y, 235R, 235D, 236D, 236N,239D, 267D, 267E, 268E, 268D, 328F, and 328Y, wherein numbering isaccording to an EU index as in Kabat. In another embodiment, the Fcdomain that binds FcγRIIb comprises at least one modification selectedfrom the group consisting of 235Y, 235R, 236D, 267D, 267E, and 328F. Ina further embodiment, the Fc domain that binds FcγRIIb comprises atleast one modification selected from 267E and 328F. In a furtherembodiment, the Fc domain that binds FcγRIIb comprises modificationsselected from 235D/267E, 235Y/267E, 235D/267D, 235I/267E, 235I/267D,235Y/267D, 236D/267E, 236D/267D, 267E/328F, 267D/328F, 268D/267E,H268D/S267D, 268E/267E, and 268E/267D. In a further embodiment, the Fcdomain that binds FcγRIIb comprises modifications selected from235D/267E, 235Y/267E, 235Y/267D, 236D/267E, 267E/328F, 268D/267E,268E/267E, and 268E/267D. In a further embodiment, the Fc domain thatbinds FcγRIIb comprises modifications 236D/267E/328F. In anotherembodiment, the Fc domain that binds FcγRIIb comprises modifications267E/328F, wherein numbering is according to the EU index as in Kabat.In some embodiments, the Fc domain that binds FcγRIIb comprises SEQ IDNO:7 and SEQ ID NO:9. In some embodiments, the Fv domain that binds CD19comprises one or more sequence selected from the group consisting of SEQID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, andSEQ ID NO:40.

As discussed above, an immunoglobulin (such as an antibody) thatcoengages FcγRIIb and CD19 can be used to treat IgG4-RD. In oneembodiment, administration of an immunoglobulin that coengages FcγRIIband CD19 to treat IgG4-RD can increase or decrease the values ofpharmacodynamics markers. In a further embodiment, administration of animmunoglobulin that coengages FcγRIIb and CD19 to treat IgG4-RD canresult in the reduction of severity and/or number of symptoms associatedwith IgG4-RD. Administration of an immunoglobulin that coengages FcγRIIband CD19 to treat IgG4-RD can also reduce the IgG4-RD responder index(IgG4-RD RI) score. IgG4-RD RI is described in more detail below. Usingan antibody that coengages FcγRIIb and CD19, the IgG4-RD RI score can besignificantly reduced relative to baseline.

Pharmacodynamic Markers

In some instances, administration of an immunoglobulin that coengagesFcγRIIb and CD19 to treat IgG4-RD can result in a change in the value ofpharmacodynamic markers. Pharmacodynamic markers may include, but arenot limited to, B cell number, plasmablast number, CD4+SLAMF7+ CTL cellnumber, and CD19 receptor occupancy. In some instances, a decrease intotal B cell number can result. In other instances, a decrease in totalplasmablast number can result. In other instances, a decrease inCD4+SLAMF7+ CTL cell number can result. In other instances, an increasein CD19 receptor occupancy can result.

A change in one or more pharmacodynamics marker may be observed within 1day of administration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 1 day ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 2 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 3 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 4 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 5 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 6 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 7 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 8 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 9 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 10 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 11 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 12 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 13 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 14 days ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 1 month ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 2 months ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 3 months ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 4 months ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 5 months ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 6 months ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 7 months ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 8 months ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 9 months ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 10 months ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 11 months ofadministration. In some instances, a change in one or morepharmacodynamics marker may be observed at about 12 months ofadministration.

Administration of an immunoglobulin that coengages FcγRIIb and CD19 canreduce the total B cell numbers in a subject with an IgG4-RD.

A reduction of B cells can observed at about 24 hours, about 2 days,about 3 days, about 4 days, about 5 days, about 6 days, about 7 days,about 8 days, about 9 days, about 10 days, about 11 days, about 12 days,about 13 days, about 14 days, about 3 weeks, about 4 weeks, about 5weeks, about 6 weeks, about 2 months, or about 3 months followingadministration of an immunoglobulin that coengages FcγRIIb and CD19.Specifically, a reduction of B cells can be observed within 7 days ofadministration of an immunoglobulin that coengages FcγRIIb and CD19.

A reduction of B cells can be determined by methods known in the art.For example, alleviation of signs or symptoms of an IgG4-RD cancorrelate with a reduction of B cells. Additionally, a reduction of Bcells can be determined by measuring the quantity of p B cells in abiological sample. A biological sample can be a tissue sample or a fluidsample. For example, microscopy or flow cytometry can be used to measurea reduction in B cells.

The B cells can be reduced by greater than 10% relative to baseline,where baseline is the amount of B cells prior to initiation oftreatment. In some variations, the B cells can be reduced by greaterthan 15% relative to baseline. In some variations, the B cells can bereduced greater than 20% relative to baseline. In some variations, the Bcells can be reduced greater than 25% relative to baseline. In somevariations, the p B cells can be reduced greater than 30% relative tobaseline. In some variations, the B cells can be reduced greater than35% relative to baseline. In some variations, the B cells can bereduced, greater than 40% relative to baseline. In some variations, theB cells can be reduced greater than 45% relative to baseline. In somevariations, the B cells can be reduced greater than 50% relative tobaseline. In some variations, the B cells can be reduced greater than55% relative to baseline. In some variations, the B cells can be reducedgreater than 60% relative to baseline. In some variations, the B cellscan be reduced greater than 65% relative to baseline. In somevariations, the B cells can be reduced greater than 70% relative tobaseline. In some variations, the B cells can be reduced greater than75% relative to baseline. In some variations, the B cells can be reducedgreater than 80% relative to baseline. In some variations, the B cellscan be reduced greater than 85% relative to baseline. In somevariations, the B cells can be reduced greater than 90% relative tobaseline. In some variations, the B cells can be reduced or greater than95% relative to baseline.

Additionally, the B cells can be reduced by greater than 1.2-foldrelative to baseline. In some variations, the B cells can be reduced bygreater than 1.5-fold relative to baseline. In some variations, the Bcells can be reduced by greater than 2-fold relative to baseline. Insome variations, the B cells can be reduced by greater than 2.5-foldrelative to baseline. In some variations, the B cells can be reduced bygreater than 5-fold relative to baseline. In some variations, the Bcells can be reduced by greater than 10-fold relative to baseline. Insome variations, the B cells can be reduced by greater than 20-foldrelative to baseline. In some variations, the B cells can be reduced bygreater than 25-fold relative to baseline. In some variations, the Bcells can be reduced by greater than 50-fold relative to baseline. Insome variations, the B cells can be reduced by greater than 75-foldrelative to baseline. In some variations, the B cells can be reduced bygreater than 100-fold relative to baseline. In some variations, the Bcells can be reduced by greater than 200-fold relative to baseline. Insome variations, the B cells can be reduced by greater than 500-foldrelative to baseline. In some variations, the B cells can be reduced byor greater than 1000-fold relative to baseline.

Administration of an immunoglobulin that coengages FcγRIIb and CD19 canreduce or deplete plasmablast numbers in a subject with an IgG4-RD. Asubject with IgG4-RD can have plasmablast levels greater than 100cells/mL. For example, a subject with IgG4-RD can have plasmablastlevels greater than 100 cells/m L, greater than 200 cells/m L, greaterthan 300 cells/m L, greater than 400 cells/mL, greater than 500cells/mL, greater than 600 cells/mL, greater than 700 cells/mL, greaterthan 800 cells/m L, greater than 900 cells/mL, greater than 1000cells/mL, greater than 2000 cells/mL, greater than 3000 cells/mL,greater than 4000 cells/mL or greater than 5000 cells/mL. Accordingly,an immunoglobulin for the treatment of an IgG4-RD can deplete or reducethe number of plasmablasts.

A reduction or depletion of plasmablasts can observed at about 24 hours,about 2 days, about 3 days, about 4 days, about 5 days, about 6 days,about 7 days, about 8 days, about 9 days, about 10 days, about 11 days,about 12 days, about 13 days, about 14 days, about 3 weeks, about 4weeks, about 5 weeks, about 6 weeks, about 2 months, or about 3 monthsfollowing administration of an immunoglobulin that coengages FcγRIIb andCD19.

A reduction or depletion of plasmablasts can be determined by methodsknown in the art. For example, alleviation of signs or symptoms of anIgG4-RD can correlate with a reduction or depletion of plasmablasts.Additionally, a reduction or depletion of plasmablasts can be determinedby measuring the quantity of plasmablasts in a biological sample. Abiological sample can be a tissue sample or a fluid sample. For example,microscopy or flow cytometry can be used to measure a reduction ordepletion in plasmablasts. The plasmablasts can be reduced or depletedby greater than 10% relative to baseline, where baseline is the amountof plasmablasts prior to initiation of treatment. In some variations,the plasmablasts can be reduced or depleted by greater than 15% relativeto baseline. In some variations, the plasmablasts can be reduced ordepleted greater than 20% relative to baseline. In some variations, theplasmablasts can be reduced or depleted greater than 25% relative tobaseline. In some variations, the plasmablasts can be reduced ordepleted greater than 30% relative to baseline. In some variations, theplasmablasts can be reduced or depleted greater than 35% relative tobaseline. In some variations, the plasmablasts can be reduced ordepleted, greater than 40% relative to baseline. In some variations, theplasmablasts can be reduced or depleted greater than 45% relative tobaseline. In some variations, the plasmablasts can be reduced ordepleted greater than 50% relative to baseline. In some variations, theplasmablasts can be reduced or depleted greater than 55% relative tobaseline. In some variations, the plasmablasts can be reduced ordepleted greater than 60% relative to baseline. In some variations, theplasmablasts can be reduced or depleted greater than 65% relative tobaseline. In some variations, the plasmablasts can be reduced ordepleted greater than 70% relative to baseline. In some variations, theplasmablasts can be reduced or depleted greater than 75% relative tobaseline. In some variations, the plasmablasts can be reduced ordepleted greater than 80% relative to baseline. In some variations, theplasmablasts can be reduced or depleted greater than 85% relative tobaseline. In some variations, the plasmablasts can be reduced ordepleted greater than 90% relative to baseline. In some variations, theplasmablasts can be reduced or depleted or greater than 95% relative tobaseline.

Additionally, the plasmablasts can be reduced by greater than 1.2-foldrelative to baseline. In some variations, the plasmablasts can bereduced by greater than 1.5-fold relative to baseline. In somevariations, the plasmablasts can be reduced by greater than 2-foldrelative to baseline. In some variations, the plasmablasts can bereduced by greater than 2.5-fold relative to baseline. In somevariations, the plasmablasts can be reduced by greater than 5-foldrelative to baseline. In some variations, the plasmablasts can bereduced by greater than 10-fold relative to baseline. In somevariations, the plasmablasts can be reduced by greater than 20-foldrelative to baseline. In some variations, the plasmablasts can bereduced by greater than 25-fold relative to baseline. In somevariations, the plasmablasts can be reduced by greater than 50-foldrelative to baseline. In some variations, the plasmablasts can bereduced by greater than 75-fold relative to baseline. In somevariations, the plasmablasts can be reduced by greater than 100-foldrelative to baseline. In some variations, the plasmablasts can bereduced by greater than 200-fold relative to baseline. In somevariations, the plasmablasts can be reduced by greater than 500-foldrelative to baseline. In some variations, the plasmablasts can bereduced by or greater than 1000-fold relative to baseline.

Administration of an immunoglobulin that coengages FcγRIIb and CD19 canreduce the CD4+ cytotoxic T lymphocyte cell (CD4+ CTL cell) numbers in asubject with an IgG4-RD. More specifically, administration of animmunoglobulin that coengages FcγRIIb and CD19 can reduce theCD4+SLAMF7+ CTL cell (effector CD4+ CTL cell) numbers in a subject withan IgG4-RD. CD4+SLAMF7+ CTL numbers are increased in the peripheralblood of IgG4-RD patients (see FIG. 29). It has been reported thatrituximab decreases circulating CD4+SLMF7+ CTL numbers in patients withIgG4-RD after rituximab therapy, but not until 1 month or more aftertherapy. (Hamid Mattoo, et al., J Allergy Clin. Immunol. 2016).Reduction of CD4+SLAMF7+CTL after administration of an immunoglobulinthat coengages FcγRIIb and CD19 occurs much more quickly, as, in someinstances, reductions can observed within 1 day of administration.

A reduction of CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can observedat about 24 hours, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 7 days, about 8 days, about 9 days, about 10days, about 11 days, about 12 days, about 13 days, about 14 days, about3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 2 months, orabout 3 months following administration of an immunoglobulin thatcoengages FcγRIIb and CD19. Specifically, a reduction of CD4+ CTL cellscan be observed within 1 days of administration of an immunoglobulinthat coengages FcγRIIb and CD19. A reduction of CD4+CD4+SLAMF7+CTL cellscan be observed within 1 days of administration of an immunoglobulinthat coengages FcγRIIb and CD19.

A reduction of CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can bedetermined by methods known in the art. For example, alleviation ofsigns or symptoms of an IgG4-RD can correlate with a reduction of CD4+CTL cells and/or CD4+SLAMF7+CTL cells. Additionally, a reduction of CD4+CTL cells and/or CD4+SLAMF7+CTL cells can be determined by measuring thequantity of p CD4+CTL cells and/or CD4+SLAMF7+CTL cells in a biologicalsample. A biological sample can be a tissue sample or a fluid sample.For example, microscopy or flow cytometry can be used to measure areduction in CD4+ CTL cells and/or CD4+SLAMF7+CTL cells.

The CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can be reduced by greaterthan 10% relative to baseline, where baseline is the amount of CD4+ CTLcells and/or CD4+SLAMF7+CTL cells prior to initiation of treatment. Insome variations, the CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can bereduced by greater than 15% relative to baseline. In some variations,the CD4+CTL cells and/or CD4+SLAMF7+CTL cells can be reduced greaterthan 20% relative to baseline. In some variations, the CD4+ CTL cellsand/or CD4+SLAMF7+CTL cells can be reduced greater than 25% relative tobaseline. In some variations, the p CD4+CTL cells and/or CD4+SLAMF7+CTLcells can be reduced greater than 30% relative to baseline. In somevariations, the CD4+ CTL cells and/or CD4+SLAMF7+ CTL cells can bereduced greater than 35% relative to baseline. In some variations, theCD4+ CTL cells and/or CD4+SLAMF7+ CTL cells can be reduced, greater than40% relative to baseline. In some variations, the CD4+ CTL cells and/orCD4+SLAMF7+CTL cells can be reduced greater than 45% relative tobaseline. In some variations, the CD4+ CTL cells and/or CD4+SLAMF7+CTLcells can be reduced greater than 50% relative to baseline. In somevariations, the CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can bereduced greater than 55% relative to baseline. In some variations, theCD4+ CTL cells and/or CD4+SLAMF7+ CTL cells can be reduced greater than60% relative to baseline. In some variations, the CD4+ CTL cells and/orCD4+SLAMF7+CTL cells can be reduced greater than 65% relative tobaseline. In some variations, the CD4+ CTL cells and/or CD4+SLAMF7+CTLcells can be reduced greater than 70% relative to baseline. In somevariations, the CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can bereduced greater than 75% relative to baseline. In some variations, theCD4+ CTL cells and/or CD4+SLAMF7+CTL cells can be reduced greater than80% relative to baseline. In some variations, the CD4+ CTL cells and/orCD4+SLAMF7+CTL cells can be reduced greater than 85% relative tobaseline. In some variations, the CD4+ CTL cells and/or CD4+SLAMF7+CTLcells can be reduced greater than 90% relative to baseline. In somevariations, the CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can bereduced or greater than 95% relative to baseline.

Additionally, the CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can bereduced by greater than 1.2-fold relative to baseline. In somevariations, the CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can bereduced by greater than 1.5-fold relative to baseline. In somevariations, the CD4+ CTL cells and/or CD4+SLAMF7+ CTL cells can bereduced by greater than 2-fold relative to baseline. In some variations,the CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can be reduced by greaterthan 2.5-fold relative to baseline. In some variations, the CD4+ CTLcells and/or CD4+SLAMF7+CTL cells can be reduced by greater than 5-foldrelative to baseline. In some variations, the CD4+ CTL cells and/orCD4+SLAMF7+CTL cells can be reduced by greater than 10-fold relative tobaseline. In some variations, the CD4+ CTL cells and/or CD4+SLAMF7+CTLcells can be reduced by greater than 20-fold relative to baseline. Insome variations, the CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can bereduced by greater than 25-fold relative to baseline. In somevariations, the CD4+CTL cells and/or CD4+SLAMF7+CTL cells can be reducedby greater than 50-fold relative to baseline. In some variations, theCD4+ CTL cells and/or CD4+SLAMF7+CTL cells can be reduced by greaterthan 75-fold relative to baseline. In some variations, the CD4+CTL cellsand/or CD4+SLAMF7+CTL cells can be reduced by greater than 100-foldrelative to baseline. In some variations, the CD4+ CTL cells and/orCD4+SLAMF7+CTL cells can be reduced by greater than 200-fold relative tobaseline. In some variations, the CD4+CTL cells and/or CD4+SLAMF7+CTLcells can be reduced by greater than 500-fold relative to baseline. Insome variations, the CD4+ CTL cells and/or CD4+SLAMF7+CTL cells can bereduced by or greater than 1000-fold relative to baseline.

Administration of an immunoglobulin that coengages FcγRIIb and CD19 canresult in CD19 receptor occupancy in a subject with an IgG4-RD. Withoutwishing to be bound by theory, an immunoglobulin of the disclosure foruse in the treatment of an IgG4-RD coengages FcγRIIb and CD19 expressedon B cells. This coengagment results in occupancy the CD19 receptor bythe immunoglobulin that coengages FcγRIIb and CD19. CD19 receptoroccupancy can be determined by methods known in the art, such as, butnot limited to, flow cytometry.

The CD19 receptor occupancy in a subject after administration of animmunoglobulin that coengages FcγRIIb and CD19 may be from about 30% toabout 100%. In some embodiments, the CD19 receptor occupancy is equal toor greater than 30%. In some embodiments, the CD19 receptor occupancy isequal to or greater than 40%. In some embodiments, the CD19 receptoroccupancy is equal to or greater than 50%. In some embodiments, the CD19receptor occupancy is equal to or greater than 60%. In some embodiments,the CD19 receptor occupancy is equal to or greater than 70%. In someembodiments, the CD19 receptor occupancy is equal to or greater than80%. In some embodiments, the CD19 receptor occupancy is equal to orgreater than 90%. In some embodiments, the CD19 receptor occupancy isequal to or greater than 95%. In some embodiments, the CD19 receptoroccupancy is 100%.

Reduction in Symptoms

In some instances, administration of an immunoglobulin that coengagesFcγRIIb and CD19 can result in a reduction in the symptoms associatedwith IgG4-RD. In some aspects, the reduction can be a reduction in theseverity (i.e., intensity) of at least one symptom. In other aspects,the reduction can be a reduction in the number of symptoms. In furtheraspects, the reduction can be a reduction in the number affected organsor tissues. In other aspects, the reduction is a combination of one ormore of a reduction in the severity (i.e., intensity) of at least onesymptom, a reduction in the number affected organs or tissues, and areduction in the number affected organs or tissues.

Symptoms are generally based upon the organ or tissue affected by theIgG4-RD. Non-limiting examples of symptoms of IgG4-RD include fibrosis(scarring), organ dysfunction, organ failure, weight loss, inflammation,pain, swelling, mass lesions, jaundice, seizures, paralysis orhemiparesis, cranial nerve palsies, sensorineural hearing loss,pituitary hormone deficiencies, loss of vision, proptosis, constrictivepericarditis, heart block, ruptured aortic aneurysm, aortic dissection,carotid artery dissection, angina, sudden cardiac death, airwayobstruction, pleural effusion, esophageal obstruction, bowelobstruction, renal failure, hydronephrosis, and testicular pain. In someaspects, a reduction in symptoms is a reduction in the number of organsand/or tissues affected. In other aspects, a reduction in symptoms is areduction in the severity of the symptoms in at least one organ ortissue.

A subject with IgG4-RD can exhibit symptoms in one or more organs. Forexample, a subject with IgG4-RD can exhibit symptoms in 1 or more, 2 ormore, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more,9 or more, or 10 or more organs. In various embodiments, a subject withIgG4-RD exhibit symptoms in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or moreorgans. In one embodiment, a subject with IgG4-RD can exhibit symptomsin 4 or more organs. Non-limiting examples of organs that exhibitIgG4-RD symptoms include, but are not limited to, lymph nodes,submandibular glands, parotid glands, lacrimal glands, kidney, heart,pericardium, orbit, nasal cavity, lungs, bile ducts, salivary glands,and pancreas. In certain embodiments, the symptomatic organs includelymph nodes, submandibular glands, parotid glands, and lacrimal glands.

A reduction in symptoms may be observed within 7 days of administration.In some instances, a reduction in symptoms can be observed at about 7days of administration of the antibody. In some instances, a reductionin symptoms can be observed at about 8 days. In some instances, areduction in symptoms can be observed at about 9 days. In someinstances, a reduction in symptoms can be observed at about 10 days. Insome instances, a reduction in symptoms can be observed at about 11days. In some instances, a reduction in symptoms can be observed atabout 12 days. In some instances, a reduction in symptoms can beobserved at about 13 days. In some instances, a reduction in symptomscan be observed at about 14 days.

In some instances, reduction in symptoms can be observed in changes insalivary glands, lacrimal glands, extra ocular muscle involvement, lymphnodes, kidney, bile duct, and/or pancreatic involvement. Reduction insymptoms in salivary glands, lacrimal glands, extra ocular muscleinvolvement, lymph nodes, kidney, bile duct, and/or pancreaticinvolvement may be observed in about 7 days. In other instances,reduction in symptoms in salivary glands, lacrimal glands, extra ocularmuscle involvement, lymph nodes, kidney, bile duct, and/or pancreaticinvolvement may be observed in about 8 days. In other instances,reduction in symptoms in salivary glands, lacrimal glands, extra ocularmuscle involvement, lymph nodes, kidney, bile duct, and/or pancreaticinvolvement may be observed in about 9 days. In other instances,reduction in symptoms in salivary glands, lacrimal glands, extra ocularmuscle involvement, lymph nodes, kidney, bile duct, and/or pancreaticinvolvement may be observed in about 10 days. In other instances,reduction in symptoms in salivary glands, lacrimal glands, extra ocularmuscle involvement, lymph nodes, kidney, bile duct, and/or pancreaticinvolvement may be observed in about 11 days. In other instances,reduction in symptoms in salivary glands, lacrimal glands, extra ocularmuscle involvement, lymph nodes, kidney, bile duct, and/or pancreaticinvolvement may be observed in about 12 days. In other instances,reduction in symptoms in salivary glands, lacrimal glands, extra ocularmuscle involvement, lymph nodes, kidney, bile duct, and/or pancreaticinvolvement may be observed in about 13 days. In other instances,reduction in symptoms in salivary glands, lacrimal glands, extra ocularmuscle involvement, lymph nodes, kidney, bile duct, and/or pancreaticinvolvement may be observed in about 14 days. In other instances,reduction in symptoms in salivary glands, lacrimal glands, extra ocularmuscle involvement, lymph nodes, kidney, bile duct, and/or pancreaticinvolvement may be observed after about 14 days.

IgG4-RD Responder Index

In some instances, administration of an immunoglobulin that coengagesFcγRIIb and CD19 can result a reduction in the IgG4-RD responder index(IgG4-RD RI) score. An IgG4-RD RI, is a tool designed to detect anychanges in disease activity and identify improvement and worsening inthe same and/or different organ systems. For a general overview of theIgG4-RI scoring methodology see Carruthers et al., International Journalof Rheumatology 2012, the disclosure of which is hereby incorporated byreference in its entirety. In brief, the IgG4-RD RI uses a scoringsystem for each organ system or site and asks a clinician to rate theextent of disease activity and damage at the time of the clinicalencounter. The following organs sites are typically rated by theclinician: pachymeninges, pituitary gland, orbits and lacrimal glands,salivary glands, thyroid, lymph nodes, lungs, aorta and large bloodvessels, retroperitoneum, mediastinum, and mesentery, pancreas, bileduct and liver, kidney, skin, and other sclerosis/mass formation. Theclinician enters a score from 0-3 for each organ/site affected,indicates whether the organ site is symptomatic (yes/no), indicateswhether the disease activity for each organ/site requires urgenttreatment (yes/no); and indicates whether organ/site dysfunction isrelated to damage rather that active disease (yes/no). For purposes ofthis disclosure, a scoring system of 0-3 was employed. Specifically, thefollowing scoring system for disease activity is employed: 0—unaffectedor resolved; 1—improved but persistent; 2—new or recurrence (while offtreatment) or unchanged; and 3—worse or new despite treatment. The sumof the disease activity in all the organ sites results in the totalactivity score. If an organ site is labeled urgent (“yes”) then thescore for that site is doubled. The IgG4-RD RI score can then becompared between assessments to determine disease activity over time andbe used for clinical trial endpoints.

The IgG4-RD RI may, in some instances as disclosed in Currthers et al.,take into account the subject's serum IgG4 concentration. For thisdisclosure the IgG4-RD RI does not take into account the subject's serumIgG4 concentrations.

The IgG4-RD RI score can be maintained relative to baseline or bereduced relative to baseline, where baseline is the IgG4-RD RI scoreprior to initiation of treatment. When the IgG4-RD RI score ismaintained, there is no change in the IgG4-RD RI score relative tobaseline.

In some instances, a reduction relative to baseline in the IgG4-RD RIscore may be observed within 14 days of administration. In someinstances, a reduction in the IgG4-RD RI score may be observed at about14 days of administration. In some instances, a reduction in the IgG4-RDRI score at about 1 month of administration. In some instances, areduction in the IgG4-RD RI score may be observed at about 2 months ofadministration. In some instances, a reduction in the IgG4-RD RI scoremay be observed at about 3 months of administration. In some instances,a reduction in the IgG4-RD RI score may be observed at about 4 months ofadministration. In some instances, a reduction in the IgG4-RD RI scoremay be observed at about 5 months of administration. In some instances,a reduction in the IgG4-RD RI score may be observed at about 6 months ofadministration. In some instances, a reduction in the IgG4-RD RI scoremay be observed at about 7 months of administration. In some instances,a reduction in the IgG4-RD RI score may be observed at about 8 months ofadministration. In some instances, a reduction in the IgG4-RD RI scoremay be observed at about 9 months of administration. In some instances,a reduction in the IgG4-RD RI score may be observed at about 10 monthsof administration. In some instances, a reduction in the IgG4-RD RIscore may be observed at about 11 months of administration. In someinstances, a reduction in the IgG4-RD RI score may be observed at about12 months of administration.

When the IgG4-RD-RI score is reduced relative to baseline, the score maydecrease by at least 1 relative to baseline. In some variations, theIgG4-RD-RI score may decrease by at least 2 relative to baseline. Insome variations, the IgG4-RD-RI score may decrease by at least 3. Insome variations, the IgG4-RD-RI score may decrease by at least 4. Insome variations, the IgG4-RD-RI score may decrease by at least 5. Insome variations, the IgG4-RD-RI score may decrease by at least 6. Insome variations, the IgG4-RD-RI score may decrease by at least 7. Insome variations, the IgG4-RD-RI score may decrease by at least 8. Insome variations, the IgG4-RD-RI score may decrease by at least 9. Insome variations, the IgG4-RD-RI score may decrease by at least 10. Insome variations, the IgG4-RD-RI score may decrease by at least 11. Insome variations, the IgG4-RD-RI score may decrease by at least 12. Insome variations, the IgG4-RD-RI score may decrease by at least 13. Insome variations, the IgG4-RD-RI score may decrease by at least 14. Insome variations, the IgG4-RD-RI score may decrease by at least 15. Insome variations, the IgG4-RD-RI score may decrease by at least 16. Insome variations, the IgG4-RD-RI score may decrease by at least 17. Insome variations, the IgG4-RD-RI score may decrease by at least 18. Insome variations, the IgG4-RD-RI score may decrease by at least 19. Insome variations, the IgG4-RD-RI score may decrease by at least 20relative to baseline. In certain embodiments, the IgG4-RD-RI score maybe reduced to 0.

When the IgG4-RD-RI score is reduced relative to baseline, the score maydecrease by at least 1 relative to baseline within 2 weeks of the firstdose. In some variations, the IgG4-RD-RI score may decrease by at least2 relative to baseline within 2 weeks of the first dose. In somevariations, the IgG4-RD-RI score may decrease by at least 3 within 2weeks of the first dose. In some variations, the IgG4-RD-RI score maydecrease by at least 4 within 2 weeks of the first dose. In somevariations, the IgG4-RD-RI score may decrease by at least 5 within 2weeks of the first dose. In some variations, the IgG4-RD-RI score maydecrease by at least 6 within 2 weeks of the first dose. In somevariations, the IgG4-RD-RI score may decrease by at least 7 within 2weeks of the first dose. In some variations, the IgG4-RD-RI score maydecrease by at least 8 within 2 weeks of the first dose. In somevariations, the IgG4-RD-RI score may decrease by at least 9 within 2weeks of the first dose. In some variations, the IgG4-RD-RI score maydecrease by at least 10 within 2 weeks of the first dose. In somevariations, the IgG4-RD-RI score may decrease by at least 11 within 2weeks of the first dose. In some variations, the IgG4-RD-RI score maydecrease by at least 12 within 2 weeks of the first dose. In somevariations, the IgG4-RD-RI score may decrease by at least 13 within 2weeks of the first dose. In some variations, the IgG4-RD-RI score maydecrease by at least 14 within 2 weeks of the first dose. In somevariations, the IgG4-RD-RI score may decrease by at least 15 within 2weeks of the first dose. In some variations, the IgG4-RD-RI score maydecrease by at least 16 within 2 weeks of the first dose. In somevariations, the IgG4-RD-RI score may decrease by at least 17 within 2weeks of the first dose. In some variations, the IgG4-RD-RI score maydecrease by at least 18 within 2 weeks of the first dose. In somevariations, the IgG4-RD-RI score may decrease by at least 19 within 2weeks of the first dose. In some variations, the IgG4-RD-RI score maydecrease by at least 20 relative to baseline within 2 weeks of the firstdose. In certain embodiments, the IgG4-RD-RI score may be reduced to 0within 2 weeks of the first dose.

Reduction in Other Therapies and Therapeutic Agents

The use of an immunoglobulin that coengages FcγRIIb and CD19 to treatIgG4-RD may result in the reduction or elimination of side effectsassociated with other therapies. Without wishing to be bound by theory,the mechanism of action of the antibody reduces off-target effectsthereby reducing side effects associated with other therapies.

Further, the targeted treatment of IgG4-RD can reduce or eliminate theneed for additional agents to treat the IgG4-RD. For example, thetargeted treatment of IgG4-RD can reduce or eliminate the need foranti-inflammatory pain reliever drugs (NSAIDs such as aspirin,ibuprofen, naproxen, or Celebrex), acetaminophen, steroids,glucocorticoids (i.e. prednisone), immunosuppressive agents (i.e.azathioprine, mycophenolate mofetil), and immunosuppressive biologics(i.e. rituximab, bortezomib) typically used to treat IgG4-RD.

In certain embodiments, the targeted treatment of IgG4-RD using anantibody that coengages FcγRIIb and CD19 can result in the tapering andeventual discontinuation of steroids. Methods of tapering steroids areknown in the art. For example, a decrease in dose can be made every 2 to3 days (e.g. 6 tablets taken on day 1 and 2, 5 tablets taken on day 3and 4, 4 tablets on day 5 and 6, 3 tablets on day 7 and 8, 2 tablets onday 9 and 10, and 1 tablet on day 11 and 12). Alternatively, a dose canbe decreased by half every 3 days.

Further, in some instances IgG4-RD can be treated by administering anantibody that coengages FcγRIIb and CD19 to a subject who is refractoryto an immunosuppressive biologic therapy (e.g. rituximab, bortezomib).Subjects that are refractory to the administered immunosuppressivebiologic therapy do not respond to a given immunosuppressive biologic,such as rituximab or bortezomib, or exhibit a therapeutic response andthen re-developed symptoms of the disease. In some instances IgG4-RD canbe treated by administering an antibody that coengages FcγRIIb and CD19to a subject who is refractory to rituximab. In some instances IgG4-RDcan be treated by administering an antibody that coengages FcγRIIb andCD19 to a subject who is refractory to bortezomib.

In some instances, IgG4-RD can be treated by administering an antibodythat coengages FcγRIIb and CD19 to subject has relapsed followingtreatment with an immunosuppressive biologic. A relapsed subject hasresponded to treatment with an immunosuppressive biologic, butre-developed symptoms of the disease. In some instances theimmunosuppressive biologic may be rituximab. In some instances theimmunosuppressive biologic may be bortezomib.

Patient Polymorphisms

A number of the receptors that may interact with the immunoglobulinsdisclosed herein are polymorphic in the human population. For a givenpatient or population of patients, the efficacy of the immunoglobulinsdisclosed herein may be affected by the presence or absence of specificpolymorphisms in proteins. For example, FcγRIIIa is polymorphic atposition 158, which is commonly either V (high affinity) or F (lowaffinity). Patients with the V/V homozygous genotype are observed tohave a better clinical response to treatment with the anti-CD20 antibodyRituxan® (rituximab), likely because these patients mount a stronger NKresponse (Dall'Ozzo et. al. (2004) Cancer Res. 64:4664-9, incorporatedentirely by reference). Additional polymorphisms include but are notlimited to FcγRIIa R131 or H131, and such polymorphisms are known toeither increase or decrease Fc binding and subsequent biologicalactivity, depending on the polymorphism. Immunoglobulins disclosedherein may bind preferentially to a particular polymorphic form of areceptor, for example FcγRIIIa 158 V, or to bind with equivalentaffinity to all of the polymorphisms at a particular position in thereceptor, for example both the 158V and 158F polymorphisms of FcγRIIIa.In one embodiment, immunoglobulins disclosed herein may have equivalentbinding to polymorphisms may be used in an antibody to eliminate thedifferential efficacy seen in patients with different polymorphisms.Such a property may give greater consistency in therapeutic response andreduce non-responding patient populations. Such variant Fc withidentical binding to receptor polymorphisms may have increasedbiological activity, such as ADCC, CDC or circulating half-life, oralternatively decreased activity, via modulation of the binding to therelevant Fc receptors. In one embodiment, immunoglobulins disclosedherein may bind with higher or lower affinity to one of thepolymorphisms of a receptor, either accentuating the existing differencein binding or reversing the difference. Such a property may allowcreation of therapeutics particularly tailored for efficacy with apatient population possessing such polymorphism. For example, a patientpopulation possessing a polymorphism with a higher affinity for aninhibitory receptor such as FcγRIIb could receive a drug containing anFc variant with reduced binding to such polymorphic form of thereceptor, creating a more efficacious drug.

In one embodiment, patients are screened for one or more polymorphismsin order to predict the efficacy of the immunoglobulins disclosedherein. This information may be used, for example, to select patients toinclude or exclude from clinical trials or, post-approval, to provideguidance to physicians and patients regarding appropriate dosages andtreatment options. For example, in patients that are homozygous orheterozygous for FcγRIIIa 158F antibody drugs such as the anti-CD20 mAb,Rituximab are minimally effective (Carton 2002 Blood 99: 754-758; Weng2003 J. Clin. Oncol. 21:3940-3947, both incorporated entirely byreference); such patients may show a much better clinical response tothe antibodies disclosed herein. In one embodiment, patients areselected for inclusion in clinical trials for an immunoglobulindisclosed herein if their genotype indicates that they are likely torespond significantly better to an immunoglobulin disclosed herein ascompared to one or more currently used immunoglobulin therapeutics. Inanother embodiment, appropriate dosages and treatment regimens aredetermined using such genotype information. In another embodiment,patients are selected for inclusion in a clinical trial or for receiptof therapy post-approval based on their polymorphism genotype, wheresuch therapy contains an immunoglobulin engineered to be specificallyefficacious for such population, or alternatively where such therapycontains an Fc variant that does not show differential activity to thedifferent forms of the polymorphism.

Also disclosed are diagnostic tests to identify patients who are likelyto show a favorable clinical response to an immunoglobulin disclosedherein, or who are likely to exhibit a significantly better responsewhen treated with an immunoglobulin disclosed herein versus one or morecurrently used immunoglobulin therapeutics. Any of a number of methodsfor determining FcγR polymorphisms in humans known in the art may beused.

Furthermore, also disclosed are prognostic tests performed on clinicalsamples such as blood and tissue samples. Such tests may assay foreffector function activity, including but not limited to ADCC, CDC,phagocytosis, and opsonization, or for killing, regardless of mechanism,of cancerous or otherwise pathogenic cells. In one embodiment, ADCCassays, such as those described previously, are used to predict, for aspecific patient, the efficacy of a given immunoglobulin disclosedherein. Such information may be used to identify patients for inclusionor exclusion in clinical trials, or to inform decisions regardingappropriate dosages and treatment regimens. Such information may also beused to select a drug that contains a particular immunoglobulin thatshows superior activity in such assay.

Formulation

Pharmaceutical compositions are contemplated wherein an immunoglobulindisclosed herein and one or more therapeutically active agents areformulated. Formulations of the immunoglobulins disclosed herein areprepared for storage by mixing said immunoglobulin having the desireddegree of purity with optional pharmaceutically acceptable carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed., 1980, incorporated entirely by reference), in theform of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, acetate, and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; sweeteners and other flavoring agents;fillers such as microcrystalline cellulose, lactose, corn and otherstarches; binding agents; additives; coloring agents; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). In one embodiment, the pharmaceuticalcomposition that comprises the immunoglobulin disclosed herein may be ina water-soluble form, such as being present as pharmaceuticallyacceptable salts, which is meant to include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts that retain the biological effectiveness of the free bases andthat are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Some embodiments include atleast one of the ammonium, potassium, sodium, calcium, and magnesiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. The formulations to be used for in vivo administration maybe sterile. This is readily accomplished by filtration through sterilefiltration membranes or other methods.

The immunoglobulins disclosed herein may also be formulated asimmunoliposomes. A liposome is a small vesicle comprising various typesof lipids, phospholipids and/or surfactant that is useful for deliveryof a therapeutic agent to a mammal. Liposomes containing theimmunoglobulin are prepared by methods known in the art, such asdescribed in Epstein et al., 1985, Proc Natl Acad Sci USA, 82:3688;Hwang et al., 1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. No.4,485,045; U.S. Pat. No. 4,544,545; and PCT WO 97/38731, allincorporated entirely by reference. Liposomes with enhanced circulationtime are disclosed in U.S. Pat. No. 5,013,556, incorporated entirely byreference. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes. Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. A chemotherapeutic agent or other therapeutically active agentis optionally contained within the liposome (Gabizon et al., 1989, JNational Cancer Inst 81:1484, incorporated entirely by reference).

The immunoglobulin and other therapeutically active agents may also beentrapped in microcapsules prepared by methods including but not limitedto coacervation techniques, interfacial polymerization (for exampleusing hydroxymethylcellulose or gelatin-microcapsules, orpoly-(methylmethacylate) microcapsules), colloidal drug delivery systems(for example, liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules), and macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed., 1980, incorporated entirely by reference. Sustained-releasepreparations may be prepared. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymer, which matrices are in the form of shaped articles, e.g. films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919, incorporatedentirely by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot® (whichare injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, andProLease® (commercially available from Alkermes), which is amicrosphere-based delivery system composed of the desired bioactivemolecule incorporated into a matrix of poly-DL-lactide-co-glycolide(PLG).

In an embodiment, the immunoglobulins disclosed herein are formulatedfor intravenous (IV) administration or subcutaneous (SC) administration.Generally, a formulation for IV or SC administration comprises theimmunoglobulin, one or more buffers, one or more tonicity modifiers, oneor more solvents, and one or more surfactants. Non-limiting examples ofbuffers include phosphate, citrate, acetate, glutamate, carbonate,tartrate, triethanolamine (TRIS), glycylglycine, histidine, glycine,lysine, arginine, and other organic acids. More specifically,non-limiting examples of buffers include HEPES sodium, MES, potassiumphosphate, potassium thiocyanate, sterilant, TAE, TBE, ammoniumsulfate/HEPES, BuffAR, sodium acetate, sodium carbonate, sodium citrate,sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodiumphosphate. Additionally, the buffer may be various hydrate forms. Forexample, the buffer may be a monohydrate, a dihydrate, a trihydrate, atetrahydate, a pentahydrate, a hexahydrate, a heptahydrate, anoctahydrate, a nonahydrate, a decahydrate, an undecahydrate, and adodecahydrate. Occasionally, a hydrate may be fractional such as ahemihydrate or a sequihydrate. Non-limiting examples of tonicitymodifies include sodium chloride, acetic acid, L-proline, dextrose,mannitol, potassium chloride, glycerin, and glycerol. Non-limitingexample of solvents include water, propylene glycol, polyethyleneglycols, ethanol, dimethyl sulfoxide, N-methyl-2-pyrrolidone,glycofurol, Solketal™, glycerol formal, acetone, tetrahydrofurfurylalcohol, diglyme, dimethyl isosorbide, and ethyl lactate. Non-limitingexamples of solvents include polysorbates (e.g. polysorbate-20,polysorbate-80), polyoxyethylene sorbitan monooleate (Tween 80),sorbitan monooleate polyoxyethylene sorbitan monolaurate (Tween 20),sorbitan trioleate (span 85), lecithin, and polyoxyethylenepolyoxypropylene copolymers (Pluronics, Pluronic F-68).

In certain embodiments, an IV formulation comprises the immunoglobulin,one or more buffers, one or more tonicity modifiers, one or moresolvents, and one or more surfactants. Specifically, the buffer can beone or more sodium phosphate buffers. For example, the buffer can besodium phosphate monobasic monohydrate, sodium phosphate dibasicheptahydrate, combinations thereof. In an embodiment, the tonicitymodifier is sodium chloride. In another embodiment, the solvent iswater. In a further embodiment, the surfactant is a polysorbate. Forexample, the polysorbate is polysorbate-20. Specifically, an IVformulation comprises the immunoglobulin, sodium phosphate monobasicmonohydrate, sodium phosphate dibasic heptahydrate, sodium chloride,water, and polysorbate-20. The amounts of immunoglobulin, buffer,tonicity modifier, solvent, and surfactants may vary. The amount ofimmunoglobulin can be about 1 mg to about 500 mg per mL or about 1 mg toabout 100 mg per mL or about 1 mg to about 50 mg per mL. The amount ofbuffer may be about 1 to about 10 mg per mL or about 1 to about 5 mg permL or about 1 to about 2.5 mg per mL. The amount of tonicity modifiermay be about 5 to about 15 mg per mL or about 5 to about 10 mg per mL orabout 8 to about 10 mg per mL. The amount of surfactant may be about0.01 mg to about 1 mg per mL or about 0.01 to about 0.5 mg per mL orabout 0.05 to about 0.2 mg per mL. Specifically, an IV formulationcomprises the immunoglobulin in an amount from about 1 mg to about 500mg per mL or about 1 mg to about 100 mg per mL or about 1 mg to about 50mg per mL, sodium phosphate monobasic monohydrate and sodium phosphatedibasic heptahydrate in an amount from about 1 to about 10 mg per mL orabout 1 to about 5 mg per mL or about 1 to about 2.5 mg per mL, sodiumchloride in an amount from about 1 to about 10 mg per mL or about 1 toabout 5 mg per mL or about 1 to about 2.5 mg per mL, water up to about 1mL, and polysorbate-20 in an amount from about 0.01 mg to about 1 mg permL or about 0.01 to about 0.5 mg per mL or about 0.05 to about 0.2 mgper mL. Specifically, an IV formulation comprises the immunoglobulin inan amount from about 1 mg to about 50 mg per mL, sodium phosphatemonobasic monohydrate and sodium phosphate dibasic heptahydrate in anamount from about 1 to about 2.5 mg per mL, sodium chloride in an amountfrom about 1 to about 2.5 mg per mL, water up to about 1 mL, andpolysorbate-20 in an amount from about 0.05 to about 0.2 mg per mL.

In certain embodiments, a SC formulation comprises the immunoglobulin,one or more buffers, one or more tonicity modifiers, one or moresolvents, and one or more surfactants. Specifically, the buffer can be asodium acetate buffer. For example, the buffer can be sodium acetatetrihydrate. In an embodiment, the tonicity modifier can be acetic acid,L-proline, and combinations thereof. In another embodiment, the solventis water. In a further embodiment, the surfactant is a polysorbate. Forexample, the polysorbate is polysorbate-80. Specifically, a SCformulation comprises the immunoglobulin, sodium acetate trihydrate,acetic acid and L-proline, water, and polysorbate-80. The amounts ofimmunoglobulin, buffer, tonicity modifier, solvent, and surfactants mayvary. The amount of immunoglobulin can be about 1 mg to about 500 mg permL or about 50 mg to about 250 mg per mL or about 100 mg to about 250 mgper mL. The amount of buffer may be about 1 to about 10 mg per mL orabout 1 to about 5 mg per mL or about 1 to about 2.5 mg per mL. Theamount of tonicity modifier may be about 5 to about 50 mg per mL orabout 10 to about 50 mg per mL or about 20 to about 40 mg per mL. Theamount of surfactant may be about 0.01 mg to about 1 mg per mL or about0.01 to about 0.5 mg per mL or about 0.05 to about 0.2 mg per mL.Specifically, a SC formulation comprises the immunoglobulin in an amountfrom about 1 mg to about 500 mg per mL or about 50 mg to about 250 mgper mL or about 100 mg to about 250 mg per mL, sodium acetate trihydratein an amount from about 1 to about 10 mg per mL or about 1 to about 5 mgper mL or about 1 to about 2.5 mg per mL, acetic acid and L-proline inan amount from about 5 to about 50 mg per mL or about 10 to about 50 mgper mL or about 20 to about 40 mg per mL, water up to about 1 mL, andpolysorbate-80 in an amount from about 0.01 mg to about 1 mg per mL orabout 0.01 to about 0.5 mg per mL or about 0.05 to about 0.2 mg per mL.Specifically, a SC formulation comprises the immunoglobulin in an amountfrom about 100 mg to about 250 mg per mL, sodium acetate trihydrate inan amount from about 1 to about 2.5 mg per mL, acetic acid and L-prolinein an amount from about 20 to about 40 mg per mL, water up to about 1mL, and polysorbate-80 in an amount from about 0.05 to about 0.2 mg permL.

Administration

Administration of the pharmaceutical composition comprising animmunoglobulin disclosed herein, e.g., in the form of a sterile aqueoussolution, may be done in a variety of ways, including, but not limitedto orally, subcutaneously, intravenously, intranasally, intraotically,transdermally, topically (e.g., gels, salves, lotions, creams, etc.),intraperitoneally, intramuscularly, intrapulmonary, vaginally,parenterally, rectally, or intraocularly. In some instances, for examplefor the treatment of wounds, inflammation, etc., the immunoglobulin maybe directly applied as a solution or spray. As is known in the art, thepharmaceutical composition may be formulated accordingly depending uponthe manner of introduction.

Subcutaneous administration may be used in circumstances where thepatient may self-administer the pharmaceutical composition. Many proteintherapeutics are not sufficiently potent to allow for formulation of atherapeutically effective dose in the maximum acceptable volume forsubcutaneous administration. This problem may be addressed in part bythe use of protein formulations comprising arginine-HCl, histidine, andpolysorbate (see WO 04091658, incorporated entirely by reference).Antibodies disclosed herein may be more amenable to subcutaneousadministration due to, for example, increased potency, improved serumhalf-life, or enhanced solubility.

As is known in the art, protein therapeutics are often delivered by IVinfusion or bolus. The antibodies disclosed herein may also be deliveredusing such methods. For example, administration may be by intravenousinfusion with 0.9% sodium chloride as an infusion vehicle.

Pulmonary delivery may be accomplished using an inhaler or nebulizer anda formulation comprising an aerosolizing agent. For example, AERx®inhalable technology commercially available from Aradigm, or Inhance™pulmonary delivery system commercially available from NektarTherapeutics may be used. Antibodies disclosed herein may be moreamenable to intrapulmonary delivery. FcRn is present in the lung, andmay promote transport from the lung to the bloodstream (e.g. Syntonix WO04004798, Bitonti et al. (2004) Proc. Nat. Acad. Sci. 101:9763-8, bothincorporated entirely by reference). Accordingly, antibodies that bindFcRn more effectively in the lung or that are released more efficientlyin the bloodstream may have improved bioavailability followingintrapulmonary administration. Antibodies disclosed herein may also bemore amenable to intrapulmonary administration due to, for example,improved solubility, or altered isoelectric point.

Furthermore, immunoglobulins disclosed herein may be more amenable tooral delivery due to, for example, improved stability at gastric pH andincreased resistance to proteolysis. Furthermore, FcRn appears to beexpressed in the intestinal epithelia of adults (Dickinson et al. (1999)J. Clin. Invest. 104:903-11, incorporated entirely by reference), soantibodies disclosed herein with improved FcRn interaction profiles mayshow enhanced bioavailability following oral administration. FcRnmediated transport of antibodies may also occur at other mucus membranessuch as those in the gastrointestinal, respiratory, and genital tracts(Yoshida et al. (2004) Immunity 20:769-83, incorporated entirely byreference).

In addition, any of a number of delivery systems are known in the artand may be used to administer the antibodies disclosed herein. Examplesinclude, but are not limited to, encapsulation in liposomes,microparticles, microspheres (e.g., PLA/PGA microspheres), and the like.Alternatively, an implant of a porous, non-porous, or gelatinousmaterial, including membranes or fibers, may be used. Sustained releasesystems may comprise a polymeric material or matrix such as polyesters,hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamicacid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-glycolicacid copolymers such as the Lupron Depot®, andpoly-D-(−)-3-hydroxyburyric acid. It is also possible to administer anucleic acid encoding an immunoglobulin disclosed herein, for example byretroviral infection, direct injection, or coating with lipids, cellsurface receptors, or other transfection agents. In all cases,controlled release systems may be used to release the immunoglobulin ator close to the desired location of action.

Dosing

The dosing amounts and frequencies of administration are, in oneembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

The concentration of the therapeutically active immunoglobulin in theformulation may vary from about 0.1 to 100 weight %. In one embodiment,the concentration of the immunoglobulin is in the range of 0.003 to 1.0molar. In order to treat a patient, a therapeutically effective dose ofthe immunoglobulin disclosed herein may be administered. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. Dosages may range from 0.0001 to 100mg/kg of body weight or greater, for example 0.1, 1, 5, 10, or 50 mg/kgof body weight. In one embodiment, dosages range from about 1 to about10 mg/kg. In another embodiment, the dosage is about 5 mg/kg.

In some embodiments, antibodies used to treat an IgG4-related diseasecan be administered at doses of greater than or equal to 0.2 mg/kg. Forexample, antibodies used to treat an IgG4-related disease can beadministered at doses of greater than or equal to 0.1 mg/kg, greaterthan or equal to 0.5 mg/kg, greater than or equal to 1 mg/kg, greaterthan or equal to 2 mg/kg, greater than or equal to 5 mg/kg, greater thanor equal to 10 mg/kg, greater than or equal to 15 mg/kg, greater than orequal to 20 mg/kg, or greater than or equal to 25 mg/kg. Alternatively,antibodies used to treat an IgG4-related disease can be administered atdoses of greater than or equal to 25 mg/kg, greater than or equal to 50mg/kg, greater than or equal to 75 mg/kg, greater than or equal to 100mg/kg, greater than or equal to 125 mg/kg, greater than or equal to 150mg/kg, greater than or equal to 175 mg/kg, or greater than or equal to200 mg/kg. In other embodiments, antibodies used to treat anIgG4-related disease can be administered at doses of about 0.2 mg/kg.For example, antibodies used to treat an IgG4-related disease can beadministered at doses of about 0.1 mg/kg, about 0.5 mg/kg, about 1mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg,about 20 mg/kg, or about 25 mg/kg. Alternatively, antibodies used totreat an IgG4-related disease can be administered at doses of about 25mg/kg, about 50 mg/kg, about 75 mg/kg, about 100 mg/kg, about 125 mg/kg,about 150 mg/kg, about 175 mg/kg, or about 200 mg/kg.

In some embodiments, only a single dose of the immunoglobulin is used.In other embodiments, multiple doses of the immunoglobulin areadministered. The elapsed time between administrations may be less than1 hour, about 1 hour, about 1-2 hours, about 2-3 hours, about 3-4 hours,about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 2-4days, about 4-6 days, about 7 days, about 14 days, or more than 14 days.In certain embodiments, the immunoglobulin is administered every 14days. In some embodiments, the immunoglobulin is administered for atleast 1 dose. In other embodiments, the immunoglobulin is administeredfor at least 2 doses. In other embodiments, the immunoglobulin isadministered for at least 3 doses. In other embodiments, theimmunoglobulin is administered for at least 4 doses. In otherembodiments, the immunoglobulin is administered for at least 5 doses. Inother embodiments, the immunoglobulin is administered for at least 6doses. In other embodiments, the immunoglobulin is administered for atleast 7 doses. In other embodiments, the immunoglobulin is administeredfor at least 8 doses. In other embodiments, the immunoglobulin isadministered for at least 9 doses. In other embodiments, theimmunoglobulin is administered for at least 10 doses. In otherembodiments, the immunoglobulin is administered for at least 11 doses.In other embodiments, the immunoglobulin is administered for at least 12doses. In other embodiments, the immunoglobulin is administered forgreater than 2 doses. In one particular embodiment, the immunoglobulinis administered every 14 days for a total of 12 doses.

In other embodiments the antibodies disclosed herein are administered inmetronomic dosing regimens, either by continuous infusion or frequentadministration without extended rest periods. Such metronomicadministration may involve dosing at constant intervals without restperiods. Typically such regimens encompass chronic low-dose orcontinuous infusion for an extended period of time, for example 1-2days, 1-2 weeks, 1-2 months, or up to 6 months or more. The use of lowerdoses may minimize side effects and the need for rest periods.

In certain embodiments the immunoglobulin disclosed herein and one ormore other prophylactic or therapeutic agents are cyclicallyadministered to the patient. Cycling therapy involves administration ofa first agent at one time, a second agent at a second time, optionallyadditional agents at additional times, optionally a rest period, andthen repeating this sequence of administration one or more times. Thenumber of cycles is typically from 2-10. Cycling therapy may reduce thedevelopment of resistance to one or more agents, may minimize sideeffects, or may improve treatment efficacy.

Combination Therapies

The antibodies disclosed herein may be administered concomitantly withone or more other therapeutic regimens or agents. The additionaltherapeutic regimes or agents may be used to improve the efficacy orsafety of the immunoglobulin. Also, the additional therapeutic regimesor agents may be used to treat the same disease or a comorbidity ratherthan to alter the action of the immunoglobulin. For example, animmunoglobulin disclosed herein may be administered to the patient alongwith chemotherapy, radiation therapy, or both chemotherapy and radiationtherapy. The immunoglobulin disclosed herein may be administered incombination with one or more other prophylactic or therapeutic agents,including but not limited to cytotoxic agents, chemotherapeutic agents,cytokines, growth inhibitory agents, anti-hormonal agents, kinaseinhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatoryagents, immunosuppressive agents, agents that promote proliferation ofhematological cells, angiogenesis inhibitors, protein tyrosine kinase(PTK) inhibitors, additional antibodies, FcγRIIb or other Fc receptorinhibitors, or other therapeutic agents.

The terms “in combination with” and “co-administration” are not limitedto the administration of said prophylactic or therapeutic agents atexactly the same time. Instead, it is meant that the immunoglobulindisclosed herein and the other agent or agents are administered in asequence and within a time interval such that they may act together toprovide a benefit that is increased versus treatment with only eitherthe immunoglobulin disclosed herein or the other agent or agents. Insome embodiments, immunoglobulins disclosed herein and the other agentor agents act additively, and sometimes synergistically. Such moleculesare suitably present in combination in amounts that are effective forthe purpose intended. The skilled medical practitioner can determineempirically, or by considering the pharmacokinetics and modes of actionof the agents, the appropriate dose or doses of each therapeutic agent,as well as the appropriate timings and methods of administration.

In some embodiments, antibodies disclosed herein for the treatment of anIgG4-related disease can be used in combination with standard treatmentsfor IgG4-RDs. Non-limiting examples of standard treatments for IgG4-RDinclude anti-inflammatory pain reliever drugs (NSAIDs such as aspirin,ibuprofen, naproxen, or Celebrex), acetaminophen, steroids,glucocorticoids (i.e. prednisone), immunosuppressive agents (i.e.azathioprine, mycophenolate mofetil), and immunosuppressive biologics(i.e. rituximab, bortezomib).

In one embodiment, the antibodies disclosed herein are administered withone or more additional molecules comprising antibodies or Fc. Theantibodies disclosed herein may be co-administered with one or moreother antibodies that have efficacy in treating the same disease or anadditional comorbidity; for example two antibodies may be administeredthat recognize two antigens that are overexpressed in a given type ofcancer, or two antigens that mediate pathogenesis of an autoimmune orinfectious disease.

Examples of anti-cancer antibodies that may be co-administered include,but are not limited to, anti-17-1A cell surface antigen antibodies suchas Panorex™ (edrecolomab); anti-4-1BB antibodies; anti-4Dc antibodies;anti-A33 antibodies such as A33 and CDP-833; anti-α4β1 integrinantibodies such as natalizumab; anti-α4β7 integrin antibodies such asLDP-02; anti-αVβ1 integrin antibodies such as F-200, M-200, and SJ-749;anti-αVβ3 integrin antibodies such as abciximab, CNTO-95, Mab-17E6, andVitaxin™; anti-complement factor 5 (C5) antibodies such as 5G1.1;anti-CA125 antibodies such as OvaRex® (oregovomab); anti-CD3 antibodiessuch as Nuvion® (visilizumab) and Rexomab; anti-CD4 antibodies such asIDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies such as Oncolysin B andOncolysin CD6; anti-CD7 antibodies such as HB2; anti-CD19 antibodiessuch as B43, MT-103, and Oncolysin B; anti-CD20 antibodies such as 2H7,2H7.v16, 2H7.v114, 2H7.v115, Bexxar0 (tositumomab, 1-131 labeledanti-CD20), Rituxan® (rituximab), and Zevalin® (Ibritumomab tiuxetan,Y-90 labeled anti-CD20); anti-CD22 antibodies such as Lymphocide™(epratuzumab, Y-90 labeled anti-CD22); anti-CD23 antibodies such asIDEC-152; anti-CD25 antibodies such as basiliximab and Zenapax®(daclizumab); anti-CD30 antibodies such as AC10, MDX-060, and SGN-30;anti-CD33 antibodies such as Mylotarg® (gemtuzumab ozogamicin),Oncolysin M, and Smart M195; anti-CD38 antibodies; anti-CD40 antibodiessuch as SGN-40 and toralizumab; anti-CD40L antibodies such as 5c8,Antova™ and IDEC-131; anti-CD44 antibodies such as bivatuzumab;anti-CD46 antibodies; anti-CD52 antibodies such as Campath®(alemtuzumab); anti-CD55 antibodies such as SC-1; anti-CD56 antibodiessuch as huN901-DM1; anti-CD64 antibodies such as MDX-33; anti-CD66eantibodies such as XR-303; anti-CD74 antibodies such as IMMU-110;anti-CD80 antibodies such as galiximab and IDEC-114; anti-CD89antibodies such as MDX-214; anti-CD123 antibodies; anti-CD138 antibodiessuch as B-B4-DM1; anti-CD146 antibodies such as AA-98; anti-CD148antibodies; anti-CEA antibodies such as cT84.66, labetuzumab, andPentacea™ anti-CTLA-4 antibodies such as MDX-101; anti-CXCR4 antibodies;anti-EGFR antibodies such as ABX-EGF, Erbitux® (cetuximab), IMC-C225,and Merck Mab 425; anti-EpCAM antibodies such as Crucell's anti-EpCAM,ING-1, and IS-IL-2; anti-ephrin B2/EphB4 antibodies; anti-Her2antibodies such as Herceptin®, MDX-210; anti-FAP (fibroblast activationprotein) antibodies such as sibrotuzumab; anti-ferritin antibodies suchas NXT-211; anti-FGF-1 antibodies; anti-FGF-3 antibodies; anti-FGF-8antibodies; anti-FGFR antibodies, anti-fibrin antibodies; anti-G250antibodies such as WX-G250 and Rencarex®; anti-GD2 gangliosideantibodies such as EMD-273063 and TriGem; anti-GD3 gangliosideantibodies such as BEC2, KW-2871, and mitumomab; anti-gpllb/IIIaantibodies such as ReoPro; anti-heparinase antibodies; anti-Her2/ErbB2antibodies such as Herceptin® (trastuzumab), MDX-210, and pertuzumab;anti-HLA antibodies such as Oncolym®, Smart 1D10; anti-HM1.24antibodies; anti-ICAM antibodies such as ICM3; anti-IgA receptorantibodies; anti-IGF-1 antibodies such as CP-751871 and EM-164;anti-IGF-1R antibodies such as IMC-A12; anti-IL-6 antibodies such asCNTO-328 and elsilimomab; anti-IL-15 antibodies such as HuMax™-IL15;anti-KDR antibodies; anti-laminin 5 antibodies; anti-Lewis Y antigenantibodies such as Hu3S193 and IGN-311; anti-MCAM antibodies; anti-Muc1antibodies such as BravaRex and TriAb; anti-NCAM antibodies such asERIC-1 and ICRT; anti-PEM antigen antibodies such as Theragyn andTherex; anti-PSA antibodies; anti-PSCA antibodies such as IG8; anti-Ptkantibodies; anti-PTN antibodies; anti-RANKL antibodies such as AMG-162;anti-RLIP76 antibodies; anti-SK-1 antigen antibodies such as MonopharmC; anti-STEAP antibodies; anti-TAG72 antibodies such as CC49-SCA andMDX-220; anti-TGF-β antibodies such as CAT-152; anti-TNF-α antibodiessuch as CDP571, CDP870, D2E7, Humira® (adalimumab), and Remicade®(infliximab); anti-TRAIL-R1 and TRAIL-R2 antibodies; anti-VE-cadherin-2antibodies; and anti-VLA-4 antibodies such as Antegren™. Furthermore,anti-idiotype antibodies including but not limited to the GD3 epitopeantibody BEC2 and the gp72 epitope antibody 105AD7, may be used. Inaddition, bispecific antibodies including but not limited to theanti-CD3/CD20 antibody Bi20 may be used.

Examples of antibodies that may be co-administered to treat autoimmuneor inflammatory disease, transplant rejection, GVHD, and the likeinclude, but are not limited to, anti-α4β7 integrin antibodies such asLDP-02, anti-beta2 integrin antibodies such as LDP-01, anti-complement(C5) antibodies such as 5G1.1, anti-CD2 antibodies such as BTI-322,MEDI-507, anti-CD3 antibodies such as OKT3, SMART anti-CD3, anti-CD4antibodies such as IDEC-151, MDX-CD4, OKT4A, anti-CD11a antibodies,anti-CD14 antibodies such as IC14, anti-CD18 antibodies, anti-CD23antibodies such as IDEC 152, anti-CD25 antibodies such as Zenapax,anti-CD40L antibodies such as 5c8, Antova, IDEC-131, anti-CD64antibodies such as MDX-33, anti-CD80 antibodies such as IDEC-114,anti-CD147 antibodies such as ABX-CBL, anti-E-selectin antibodies suchas CDP850, anti-gpIIb/IIIa antibodies such as ReoPro/Abcixima,anti-ICAM-3 antibodies such as ICM3, anti-ICE antibodies such as VX-740,anti-FcγR1 antibodies such as MDX-33, anti-IgE antibodies such asrhuMab-E25, anti-IL-4 antibodies such as SB-240683, anti-IL-5 antibodiessuch as SB-240563, SCH55700, anti-IL-8 antibodies such as ABX-IL8,anti-interferon gamma antibodies, and anti-TNFa antibodies such asCDP571, CDP870, D2E7, Infliximab, MAK-195F, anti-VLA-4 antibodies suchas Antegren. Examples of other Fc-containing molecules that may beco-administered to treat autoimmune or inflammatory disease, transplantrejection, GVHD, and the like include, but are not limited to, the p75TNF receptor/Fc fusion Enbrel® (etanercept) and Regeneron's IL-1 trap.

Examples of antibodies that may be co-administered to treat infectiousdiseases include, but are not limited to, anti-anthrax antibodies suchas ABthrax, anti-CMV antibodies such as CytoGam and sevirumab,anti-cryptosporidium antibodies such as CryptoGAM, Sporidin-G,anti-helicobacter antibodies such as Pyloran, anti-hepatitis Bantibodies such as HepeX-B, Nabi-HB, anti-HIV antibodies such asHRG-214, anti-RSV antibodies such as felvizumab, HNK-20, palivizumab,RespiGam, and anti-staphylococcus antibodies such as Aurexis, Aurograb,BSYX-A110, and SE-Mab.

Alternatively, the antibodies disclosed herein may be co-administered orwith one or more other molecules that compete for binding to one or moreFc receptors. For example, co-administering inhibitors of the inhibitoryreceptor FcγRIIb may result in increased effector function. Similarly,co-administering inhibitors of the activating receptors such as FcγRIIIamay minimize unwanted effector function. Fc receptor inhibitors include,but are not limited to, Fc molecules that are engineered to act ascompetitive inhibitors for binding to FcγRIIb FcγRIIIa, or other Fcreceptors, as well as other immunoglobulins and specifically thetreatment called IVIg (intravenous immunoglobulin). In one embodiment,the inhibitor is administered and allowed to act before theimmunoglobulin is administered. An alternative way of achieving theeffect of sequential dosing would be to provide an immediate releasedosage form of the Fc receptor inhibitor and then a sustained releaseformulation of an immunoglobulin disclosed herein. The immediate releaseand controlled release formulations could be administered separately orbe combined into one unit dosage form. Administration of an FcγRIIbinhibitor may also be used to limit unwanted immune responses, forexample anti-Factor VIII antibody response following Factor VIIIadministration to hemophiliacs.

In one embodiment, the antibodies disclosed herein are administered witha chemotherapeutic agent. By “chemotherapeutic agent” as used herein ismeant a chemical compound useful in the treatment of cancer. Examples ofchemotherapeutic agents include but are not limited to alkylating agentssuch as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates suchas busulfan, improsulfan and piposulfan; androgens such as calusterone,dromostanolone propionate, epitiostanol, mepitiostane, testolactone;anti-adrenals such as aminoglutethimide, mitotane, trilostane;anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; antibiotics such as aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, calicheamicin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti estrogens includingfor example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, andtoremifene (Fareston); anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; folic acidreplenisher such as frolinic acid; nitrogen mustards such aschlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; platinum analogs such ascisplatin and carboplatin; vinblastine; platinum; proteins such asarginine deiminase and asparaginase; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes,e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.)and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France);topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (suchas Tomudex); additional chemotherapeutics including aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;difluoromethylornithine (DMFO); elformithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; retinoic acid; esperamicins; capecitabine.Pharmaceutically acceptable salts, acids or derivatives of any of theabove may also be used.

A chemotherapeutic or other cytotoxic agent may be administered as aprodrug. By “prodrug” as used herein is meant a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active parent form.See, for example Wilman, 1986, Biochemical Society Transactions, 615thMeeting Belfast, 14:375-382; Stella et al., “Prodrugs: A ChemicalApproach to Targeted Drug Delivery,” Directed Drug Delivery; andBorchardt et al., (ed.): 247-267, Humana Press, 1985, all incorporatedentirely by reference. The prodrugs that may find use withimmunoglobulins disclosed herein include but are not limited tophosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, beta-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for use withthe antibodies disclosed herein include but are not limited to any ofthe aforementioned chemotherapeutic agents.

A variety of other therapeutic agents may find use for administrationwith the antibodies disclosed herein. In one embodiment, theimmunoglobulin is administered with an anti-angiogenic agent. By“anti-angiogenic agent” as used herein is meant a compound that blocks,or interferes to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule or aprotein, for example an antibody, Fc fusion, or cytokine, that binds toa growth factor or growth factor receptor involved in promotingangiogenesis. In one embodiment, an anti-angiogenic factor may be anantibody that binds to Vascular Endothelial Growth Factor (VEGF). Otheragents that inhibit signaling through VEGF may also be used, for exampleRNA-based therapeutics that reduce levels of VEGF or VEGF-R expression,VEGF-toxin fusions, Regeneron's VEGF-trap, and antibodies that bindVEGF-R. In an alternate embodiment, the antibody is administered with atherapeutic agent that induces or enhances adaptive immune response, forexample an antibody that targets CTLA-4. Additional anti-angiogenesisagents include, but are not limited to, angiostatin (plasminogenfragment), antithrombin III, angiozyme, ABT-627, Bay 12-9566, benefin,bevacizumab, bisphosphonates, BMS-275291, cartilage-derived inhibitor(CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatinA-4, endostatin (collagen XVIII fragment), farnesyl transferaseinhibitors, fibronectin fragment, gro-beta, halofuginone, heparinases,heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin(hCG), IM-862, interferon alpha, interferon beta, interferon gamma,interferon inducible protein 10 (IP-10), interleukin-12, kringle 5(plasminogen fragment), marimastat, metalloproteinase inhibitors (e.g.TIMPs), 2-methodyestradiol, MMI 270 (CGS 27023A), plasminogen activiatorinhibitor (PAI), platelet factor-4 (PF4), prinomastat, prolactin 16 kDafragment, proliferin-related protein (PRP), PTK 787/ZK 222594,retinoids, solimastat, squalamine, SS3304, SU5416, SU6668, SU11248,tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1(TSP-1), TNP-470, transforming growth factor beta (TGF-β),vasculostatin, vasostatin (calreticulin fragment), ZS6126, and ZD6474.

In one embodiment, the immunoglobulin is administered with a tyrosinekinase inhibitor. By “tyrosine kinase inhibitor” as used herein is meanta molecule that inhibits to some extent tyrosine kinase activity of atyrosine kinase. Examples of such inhibitors include but are not limitedto quinazolines, such as PD 153035, 4-(3-chloroanilino) quinazoline;pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo(2,3-d) pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lambert); antisensemolecules (e.g. those that bind to ErbB-encoding nucleic acid);quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering A G);pan-ErbB inhibitors such as C1-1033 (Pfizer); Affinitac (ISIS 3521;Isis/Lilly); Imatinib mesylate (ST1571, Gleevec®; Novartis); PKI 166(Novartis); GW2016 (Glaxo SmithKline); C1-1033 (Pfizer); EKB-569(Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1C11 (Imclone); or as described in any ofthe following patent publications: U.S. Pat. No. 5,804,396; PCT WO99/09016 (American Cyanimid); PCT WO 98/43960 (American Cyanamid); PCTWO 97/38983 (Warner-Lambert); PCT WO 99/06378 (Warner-Lambert); PCT WO99/06396 (Warner-Lambert); PCT WO 96/30347 (Pfizer, Inc.); PCT WO96/33978 (AstraZeneca); PCT WO96/3397 (AstraZeneca); PCT WO 96/33980(AstraZeneca), gefitinib (IRESSA™, ZD1839, AstraZeneca), and OSI-774(Tarceva™, OSI Pharmaceuticals/Genentech), all patent publicationsincorporated entirely by reference.

In another embodiment, the immunoglobulin is administered with one ormore immunomodulatory agents. Such agents may increase or decreaseproduction of one or more cytokines, up- or down-regulate self-antigenpresentation, mask MHC antigens, or promote the proliferation,differentiation, migration, or activation state of one or more types ofimmune cells. Immunomodulatory agents include but not limited to:non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin,ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin,ketoralac, oxaprozin, nabumentone, sulindac, tolmentin, rofecoxib,naproxen, ketoprofen, and nabumetone; steroids (e.g. glucocorticoids,dexamethasone, cortisone, hydroxycortisone, methylprednisolone,prednisone, prednisolone, trimcinolone, azulfidineicosanoids such asprostaglandins, thromboxanes, and leukotrienes; as well as topicalsteroids such as anthralin, calcipotriene, clobetasol, and tazarotene);cytokines such as TGFb, IFNa, IFNb, IFNg, IL-2, IL-4, IL-10; cytokine,chemokine, or receptor antagonists including antibodies, solublereceptors, and receptor-Fc fusions against BAFF, B7, CCR2, CCR5, CD2,CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28,CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, CD152,complement factors (C5, D) CTLA4, eotaxin, Fas, ICAM, ICOS, IFNα, IFNβ,IFNγ, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA-1, LFA-3,MHC, selectins, TGFβ, TNFα, TNFβ, TNF-R1, T-cell receptor, includingEnbrel® (etanercept), Humira® (adalimumab), and Remicade® (infliximab);heterologous anti-lymphocyte globulin; other immunomodulatory moleculessuch as 2-amino-6-aryl-5 substituted pyrimidines, anti-idiotypicantibodies for MHC binding peptides and MHC fragments, azathioprine,brequinar, bromocryptine, cyclophosphamide, cyclosporine A,D-penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold,hydroxychloroquine, leflunomide, malononitriloamides (e.g. leflunomide),methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin,and sulfasasazine.

In an alternate embodiment, immunoglobulins disclosed herein areadministered with a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture,and biologically active equivalents of the native sequence cytokines.

In one embodiment, cytokines or other agents that stimulate cells of theimmune system are co-administered with the immunoglobulin disclosedherein. Such a mode of treatment may enhance desired effector function.For example, agents that stimulate NK cells, including but not limitedto IL-2 may be co-administered. In another embodiment, agents thatstimulate macrophages, including but not limited to C5a, formyl peptidessuch as N-formyl-methionyl-leucyl-phenylalanine (Beigier-Bompadre et al.(2003) Scand. J. Immunol. 57: 221-8, incorporated entirely byreference), may be co-administered. Also, agents that stimulateneutrophils, including but not limited to G-CSF, GM-CSF, and the likemay be administered. Furthermore, agents that promote migration of suchimmunostimulatory cytokines may be used. Also additional agentsincluding but not limited to interferon gamma, IL-3 and IL-7 may promoteone or more effector functions.

In an alternate embodiment, cytokines or other agents that inhibiteffector cell function are co-administered with the immunoglobulindisclosed herein. Such a mode of treatment may limit unwanted effectorfunction.

In an additional embodiment, the immunoglobulin is administered with oneor more antibiotics, including but not limited to: aminoglycosideantibiotics (e.g. apramycin, arbekacin, bambermycins, butirosin,dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,ribostamycin, sisomycin, spectrinomycin), aminocyclitols (e.g.sprctinomycin), amphenicol antibiotics (e.g. azidamfenicol,chloramphenicol, florfrnicol, and thiamphemicol), ansamycin antibiotics(e.g. rifamide and rifampin), carbapenems (e.g. imipenem, meropenem,panipenem); cephalosporins (e.g. cefaclor, cefadroxil, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide,cefpirome, cefprozil, cefuroxine, cefixime, cephalexin, cephradine),cephamycins (cefbuperazone, cefoxitin, cefminox, cefmetazole, andcefotetan); lincosamides (e.g. clindamycin, lincomycin); macrolide (e.g.azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin,tobramycin), monobactams (e.g. aztreonam, carumonam, and tigernonam);mupirocin; oxacephems (e.g. flomoxef, latamoxef, and moxalactam);penicillins (e.g. amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, bexzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamecillin, penethamatehydriodide, penicillin o-benethamine, penicillin O, penicillin V,penicillin V benzoate, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium); polypeptides (e.g. bacitracin, colistin,polymixin B, teicoplanin, vancomycin); quinolones (amifloxacin,cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, feroxacin, flumequine,gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin,pipemidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin,tosufloxacin, trovafloxacin); rifampin; streptogramins (e.g.quinupristin, dalfopristin); sulfonamides (sulfanilamide,sulfamethoxazole); tetracyclenes (chlortetracycline, demeclocyclinehydrochloride, demethylchlortetracycline, doxycycline, duramycin,minocycline, neomycin, oxytetracycline, streptomycin, tetracycline,vancomycin).

Anti-fungal agents such as amphotericin B, ciclopirox, clotrimazole,econazole, fluconazole, flucytosine, itraconazole, ketoconazole,niconazole, nystatin, terbinafine, terconazole, and tioconazole may alsobe used.

Antiviral agents including protease inhibitors, reverse transcriptaseinhibitors, and others, including type I interferons, viral fusioninhibitors, and neuramidase inhibitors, may also be used. Examples ofantiviral agents include, but are not limited to, acyclovir, adefovir,amantadine, amprenavir, clevadine, enfuvirtide, entecavir, foscarnet,gangcyclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin,rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, andzidovudine, may be used.

The immunoglobulins disclosed herein may be combined with othertherapeutic regimens. For example, in one embodiment, the patient to betreated with an immunoglobulin disclosed herein may also receiveradiation therapy. Radiation therapy can be administered according toprotocols commonly employed in the art and known to the skilled artisan.Such therapy includes but is not limited to cesium, iridium, iodine, orcobalt radiation. The radiation therapy may be whole body irradiation,or may be directed locally to a specific site or tissue in or on thebody, such as the lung, bladder, or prostate. Typically, radiationtherapy is administered in pulses over a period of time from about 1 to2 weeks. The radiation therapy may, however, be administered over longerperiods of time. For instance, radiation therapy may be administered topatients having head and neck cancer for about 6 to about 7 weeks.Optionally, the radiation therapy may be administered as a single doseor as multiple, sequential doses. The skilled medical practitioner candetermine empirically the appropriate dose or doses of radiation therapyuseful herein. In accordance with another, an immunoglobulin disclosedherein and one or more other anti-cancer therapies are employed to treatcancer cells ex vivo. It is contemplated that such ex vivo treatment maybe useful in bone marrow transplantation and particularly, autologousbone marrow transplantation. For instance, treatment of cells ortissue(s) containing cancer cells with immunoglobulin and one or moreother anti-cancer therapies, such as described above, can be employed todeplete or substantially deplete the cancer cells prior totransplantation in a recipient patient.

It is of course contemplated that the antibodies disclosed herein mayemploy in combination with still other therapeutic techniques such assurgery or phototherapy.

EXAMPLES

Examples are provided below are for illustrative purposes only. Theseexamples are not meant to constrain any embodiment disclosed herein toany particular application or theory of operation.

Example 1. Methods for Inhibiting FcγRIIb⁺ Cells

FcγRIIb is expressed on a variety of immune cells, including B cells,plasma cells, dendritic cells, monocytes, and macrophages, where itplays a critical role in immune regulation. In its normal role on Bcells, FcγRIIb serves as a feedback mechanism to modulate B cellactivation through the B cell receptor (BCR). Engagement of B cellantigen receptor (BCR) by immune complexed antigen on mature B cellsactivates an intracellular signaling cascade, including calciummobilization, which leads to cell proliferation and differentiation.However, as IgG antibodies with specificity to the antigen are produced,the associated immune complexes (ICs) can crosslink the BCR withFcγRIIb, whereupon the activation of BCR is inhibited by engagement ofFcγRIIb and associated intracellular signaling pathways that interferewith the downstream pathways of BCR activation.

B cells function not only to produce antibodies and cytokines thatcontrol immune response, they are also antigen presenting cells (APCs).Internalization of antigen by BCR into a B cell can play a role inpresentation to and activation of T cells. Regulation of B cellactivation through the BCR is also potentially regulated by antibodyengagement of FcγRIIb. Other APCs such as dendritic cells, macrophages,and monocytes, are capable of internalizing antibody-bound antigenthrough activating receptors such as FcγRIIa, FcγRIIIa, and FcγRI.Expression of FcγRIIb on these cell types, particularly dendritic cells,can inhibit activation of these cell types and subsequent presentationto and activation of T cells (Desai et al., 2007, J Immunol).

A strategy for inhibiting activation of the aforementioned cell types itto use a single immunoglobulin to coengage FcγRIIb with surface antigenpresent on the FcγRIIb+ cell. In the case of B cells, based on thenatural biological mechanism, this would potentially involve dualtargeting of FcγRIIb and BCR, with the goal of mimicking immunecomplex-mediated suppression of B cell activation. FIG. 3 illustratesone such potential mechanism, in which an antibody is used to coengageboth FcγRIIb via its Fc region, and a target antigen associated with BCRcomplex, in this example CD19, via its Fv region.

Example 2. Engineering Immunoglobulins with Selectively EnhancedAffinity for FcγRIIb

Under physiological conditions, bridging of the BCR with FcγRIIb andsubsequent B cell suppression occurs via immune complexes of IgGs andcognate antigen. The design strategy was to reproduce this effect usinga single crosslinking antibody. Human IgG binds human FcγRIIb with weakaffinity (approximately 1 μM for IgG1), and FcγRIIb-mediated inhibitionoccurs in response to immune-complexed but not monomeric IgG. It wasreasoned that increasing Fc affinity to this receptor would be requiredfor maximal inhibition of B cell activation. Protein engineering methodswere used to design and screen Fc variants for enhanced FcγRIIb binding.

Using solved structures of the human Fc/FcγRIIIb complex (and thesequences of the human FcγRs, structural and sequence analysis were usedto identify FcγR positions that contribute to FcγRIIb affinity andselectivity relative to the activating receptors. The design strategyemployed two steps. First, FcγR positions that are determinants ofFcγRIIb and FcγRIIIa binding selectivity were identified by accountingfor proximity to the FcγR/Fc interface and amino acid dissimilaritybetween FcγRIIb and FcγRIIIa. Second, sequence positions in the Fcregion proximal to these FcγR positions were identified. Fc variantswere designed that incorporate substitutions at these positions.

A library of Fc variants was generated and screened to explore aminoacid modifications at these positions (see U.S. Pat. No. 8,063,187 toChu et al, the disclosures of which are incorporated by reference intheir entirety). Variants were generated and screened in the context ofan antibody targeting the antigen CD19, a regulatory component of theBCR coreceptor complex. The Fv region of the antibody is a humanized andaffinity matured version of antibody 4G7, and is referred to herein asHuAM4G7. The amino acid sequences of this antibody are provided in FIG.14A-FIG. 14D. The Fv genes for this antibody were subcloned into themammalian expression vector pTT5 (National Research Council Canada).Mutations in the Fc domain were introduced using site-directedmutagenesis (QuikChange, Stratagene, Cedar Creek, Tex.). In addition,control knock out variants with ablated affinity for Fc receptors weregenerated that comprise the substitution L328R, and either a G236Rsubstitution or an Arg inserted after position 236. These variants(G236R/L328R and ̂236R/L328R) are referred to as Fc-KO or FcγR knockout.To serve as non-CD19 Fc isotype controls, anti-respiratory syncytialvirus (RSV) and anti-FITC antibodies were constructed in the pTT5 vectorby fusing the appropriate V_(L) and V_(H) regions to the C_(C)K andC_(H)1-3 domains with Fc changes. Heavy and light chain constructs werecotransfected into HEK293E cells for expression, and antibodies werepurified using protein A affinity chromatography (Pierce Biotechnology,Rockford, Ill.).

Human Fc receptor proteins FcγRI and FcγRIIb for binding and competitionstudies were obtained from R&D Systems (Minneapolis, Minn.). Genesencoding FcγRIIa and FcγRIIIa receptor proteins were obtained from theMammalian Gene Collection (ATCC), and subcloned into pTT5 vector(National Research Council Canada) containing 6×His and GST-tags.Allelic forms of the receptors (H131 and R131 for FcγRIIa and V158 andF158 for FcγRIIIa) were generated using QuikChange mutagenesis. Vectorsencoding the receptors were transfected into HEK293T cells, and proteinswere purified using nickel affinity chromatography.

Variants were screened for receptor affinity using Biacore™ technology,also referred to as Biacore herein, a surface plasmon resonance (SPR)based technology for studying biomolecular interactions in real time.SPR measurements were performed using a Biacore 3000 instrument(Biacore, Piscataway, N.J.). A protein A/G (Pierce Biotechnology) CM5biosensor chip (Biacore) was generated using a standard primary aminecoupling protocol. All measurements were performed using HBS-EP buffer(10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% vol/vol surfactantP20, Biacore). Antibodies at 20 nM or 50 nM in HBS-EP buffer wereimmobilized on the protein A/G surface and FcγRs were injected. Aftereach cycle, the surface was regenerated by injecting glycine buffer (10mM, pH 1.5). Data were processed by zeroing time and response before theinjection of FcγR and by subtracting appropriate nonspecific signals(response of reference channel and injection of running buffer). Kineticanalyses were performed by global fitting of binding data with a 1:1Langmuir binding model using BIAevaluation software (Biacore).

A representative set of sensorgrams for binding of select variantanti-CD19 antibodies to FcγRIIb is shown in FIG. 4.

A useful quantity for analysis of the variants is their fold affinityrelative to WT IgG1, which is generated by dividing the Kd for bindingof WT IgG1 by the Kd for binding of variant for each receptor. Thesefold affinity results are provided in FIGS. 5A-5D. A number of variantshave FcγRIIb binding enhancements over 2 logs, and substantially reducedor ablated affinities for the activating receptors. In particular, S267E(single substitution) as well as L235Y/S267E, G236D/S267E, S239D/S267E,S267E/H268E, and S267E/L328F (double substitutions) have markedly higheraffinity for FcγRIIb. In addition, these variant have affinity for theactivating receptor FcγRIIIa that is either comparable to native IgG1,modestly enhanced, or even significantly reduced.

The variant with the highest affinity for FcγRIIb, S267E/L328F, showsover 2 orders of magnitude improvement in affinity to FcγRIIb, andsignificantly reduced affinity to the activating receptors, includingFcγRIIIa, FcγRI, and H131 FcγRIIa.

In order to validate the Biacore data and evaluate receptor binding ofthe variants on the cell surface, binding of select antibodies to cellsexpressing FcγRIIb was measured. Since HEK293T cells do not express CD19or FcγRs, transfection of these cells with FcγRIIb allowed an analysisof antibody binding to Fc receptors in an isolated system on a cellsurface. HEK293T cells in DMEM with 10% FBS were transfected with humanFcγRIIb cDNA in pCMV6 expression vector (Origene Technologies,Rockville, Md.), cultured for 3 days, harvested, washed twice in PBS,resuspended in PBS with 0.1% BSA (PBS/BSA), and aliquoted at 2×105 cellsper well into 96-well microtiter plates. Fc variant antibodies wereserially diluted in PBS/BSA then added to the cells and incubated withmixing for 1 h at room temperature. After extensive washing withPBS/BSA, phycoerythrin (PE)-labeled anti-human-Fab-specific goat F(ab′)2fragment was added for detection. Cells were incubated for 30 min atroom temperature, washed, and resuspended in PBS/BSA. Binding wasevaluated using a FACSCanto II flow cytometer (BD Biosciences, San Jose,Calif.), and the mean fluorescence intensity (MFI) was plotted as afunction of antibody concentration using GraphPad Prism software(GraphPad Software, San Diego, Calif.) from which half-maximal binding(EC50) values were determined by sigmoidal dose response modeling.

Receptor expression levels were assessed prior to binding of antibodies,and half-maximal effective concentration (EC50) values of the MFI atdifferent antibody concentrations were determined. FIG. 6 shows theresults of this experiment. The EC50 values of the variants testedshowed a similar rank order as the Biacore results. The cell-surfacebinding confirmed that the S267E/L328F variant of those tested has thehighest affinity for FcγRIIb, with an EC50 approximately 320-foldrelative to WT IgG1. The strong agreement between these cell surfacebinding data and the Biacore binding data support the accuracy of theaffinity measurements.

Although the variants were screened in the context of human IgG1, it iscontemplated that the variants could be used in the context of otherantibody isotypes, for example including but not limited to human IgG2,human IgG3, and human IgG4 (FIG. 1). In order to explore thetransferability of the variants to other antibody isotypes, theS267E/L328F variant was constructed and tested in the context of aIgG1/2 ELLGG antibody, which is a variant of an IgG2 Fc region (US Pub.No. 2006/0134105, herein expressly incorporated by reference). Themutations were constructed, antibodies purified, and binding datacarried out as described above. FIG. 7 shows affinities of the IgG1 andIgG1/2 variant antibodies to the human FcγRs as determined by Biacore.The data indicate that the greatly enhanced FcγRIIb affinity and theoverall FcγR binding profile are maintained in the variant IgG2 Fcregion, thus supporting the use of the variants in other isotypecontexts.

Collectively, the above data indicate that variants having specificsubstitutions provide the targeted properties, namely enhanced affinityfor FcγRIIb, and selectively enhanced FcγRIIb affinity relative to theactivating receptors FcγRI, FcγRIIa, and FcγRIIIa. Substitutions toenhance affinity to FcγRIIb include: 234, 235, 236, 239, 267, 268, and328.

Non-limiting combinations of positions for making substitution toenhance affinity to FcγRIIb include: 235/267, 236/267, 236/267, 267/328,and 268/267. Substitutions for enhancing affinity to FcγRIIb include:L234W, L235I, L235Y, L235R, L235D, G236D, G236N, S239D, S267D, S267E,H268E, H268D, L328F, and L328Y. Combinations of substitutions forenhancing affinity to FcγRIIb include: L235D/S267E, L235Y/S267E,L235D/S267D, L235I/S267E, L235I/S267D, L235Y/S267D, G236D/S267E,G236D/S267D, S267E/L328F, S267D/L328F, H268D/S267E, H268D/S267D,H268E/S267E, H268E/S267D, and G236D/S267E/L328F.

Example 3. Immunoglobulins Inhibit BCR-Mediated Primary Human B CellViability

Although normal B cells have a long in vivo half-life of approximatelyfive weeks, their lifespan is greatly reduced in vitro. BCR stimulationby crosslinking antibodies such as anti-IgM or anti-CD79b counteractsthis in vitro predisposition towards apoptosis, leading to B cellactivation and increased B cell viability. To demonstrate this, anATP-dependent B cell viability assay was performed. Human peripheralblood mononuclear cells (PBMCs) were purified from leukapheresis ofanonymous healthy volunteers (HemaCare, Van Nuys, CA) using Ficoll-PaquePlus density gradients (Amersham Biosciences, Newark, N.J.). Primaryhuman B cells were purified from PBMCs using a B cell enrichment kit(StemCell Technologies, Vancouver, British Columbia). Murine anti-humanCD79b (clone SN8) was purchased from Santa Cruz Biotechnology (SantaCruz, Calif.). Polyclonal anti-mu F(ab′)2 was purchased from Jacksonlmmunoresearch Lab (West Grove, Pa.). Anti-mu or anti-CD79b antibodyserial dilutions were performed in triplicate in 96-well microtiterplates containing RPM11640 with 10% FBS. Purified primary human B cells(5-7.5×104 per well) were added to a final volume of 100 μl, andincubated at 37° C. for 3 days. ATP-dependent luminescence wasquantified to determine cell viability (Cell Titer-Glo Cell ViabilityAssay, Promega, Madison, Wis.) and a Topcount luminometer (PerkinElmer,Waltham, Mass.) was used for data acquisition. FIGS. 15A and 15B showthe results of the assay, demonstrating the survival of primary human Bcells upon BCR activation, here carried out by crosslinking with anti-mu(A) or anti-CD79b (B) antibodies. In vivo such activation would occurvia immune complexed antigen, which for example could be an infectiousagent, or in the cause of an autoimmune or allergic reaction could be anautoimmune antigen or allergen.

The ATP-dependent luminescence assay was used to examine if BCRactivation-mediated viability of primary human B cells could besuppressed by an anti-CD19 antibody having enhanced Fc affinity forFcγRIIb. The above experiment was repeated, except that antibody serialdilutions of WT, variant, and control antibodies were performed intriplicate in 96-well microtiter plates containing RPM11640 with 10%FBS, plus anti-CD79b at 1 μg/ml to stimulate BCR. The results are shownin FIG. 9. Again, B cells possessed low viability in the absence of BCRcrosslinking, and addition of 10 μg/ml anti-CD79b antibody stimulatedsurvival by about 6-fold (cells alone vs. anti-CD79b).Anti-CD19-S267E/L328F, the variant with the highest FcγRIIb affinity,inhibited BCR-stimulated viability in a dose-dependent manner. Incontrast, control antibodies including anti-CD19-IgG1 (Fv control) andanti-RSV-S267E/L328F (Fc control) minimally suppressed viability. Toassess if this inhibitory effect required coengagement of CD19 andFcγRIIb, as opposed to simultaneous binding of each receptor bydifferent antibodies, the anti-CD19-S267E/L328F variant was compared toa combination of anti-CD19-IgG1 and anti-RSV-S267E/L328F controls atequal concentrations. The combination of these antibodies shouldsimultaneously bind to both CD19 and FcγRIIb but, unlikeanti-CD19-S267E/L328F, is unable to crosslink these receptors. As shownin FIG. 9, the combination failed to suppress BCR activation-inducedsurvival, indicating that coengagement of FcγRIIb and CD19 by a singlemolecule is required to inhibit BCR-mediated viability.

Example 4. Immunoglobulins Inhibit BCR-Dependent Anti-Apoptotic Effectin Primary Human B Cells

Although normal B cells in vivo have a long half-life of approximately˜5 weeks, in vitro this lifespan is greatly reduced, with increasedapoptosis due to the lack of appropriate niche. B cell activation viastimulation via the BCR induces an anti-apoptotic effect and prolongsviability, as demonstrated in FIG. 8. In order to determine whether theantiproliferative activity of the IIbE variant was a result ofneutralizing BCR-mediated survival signals, thereby allowing in vitroapoptosis to proceed, an annexin-V staining assay was performed. 1×105purified primary human B cells were incubated for 24 h at 37° C. intriplicate with 1 μg/ml anti-CD79b and serial dilutions of anti-CD19 orcontrol antibodies in 100 μl RPM11640 with 10% FBS. After incubation,cells were harvested and stained with PE-conjugated annexin-V(Biovision, Mountain View, Calif.) and 7-amino-actinomycin D (7-AAD,Invitrogen, Carlsbad, Calif.) at 5 μg/ml. Theannexin-V-positive/7-AAD-negative cells were acquired using a FACSCantoII flow cytometer, and analyzed with FACSDiva 5 analysis software (BDBiosciences).

The data are shown in FIG. 10. Annexin-V staining of primary human Bcells cultured in the presence or absence of anti-CD79b confirmed thatapoptosis was suppressed by BCR activation (FIG. 10, cells alone vs.anti-CD79b). This survival signal was neutralized in a dose-dependentmanner by anti-CD19-S267E/L328F, but not by anti-RSV-S267E/L328F Fccontrol or anti-CD19-IgG1 Fv control antibodies. Inhibition of theanti-apoptotic effect, like inhibition of calcium mobilization and cellproliferation, requires coengagement of CD19 and FcγRIIb by a singleantibody, because the combination of anti-CD19-IgG1 andanti-RSV-S267E/L328F (Fv and Fc controls, respectively) did notstimulate apoptosis. These data indicate that the anti-CD19 IIbE variantinhibits BCR-induced B cell proliferation by suppressing anti-apoptoticsurvival signals.

Example 5. In Vivo Data Demonstrating Potential for Treating Autoimmuneor Inflammatory Disorder

A hallmark of autoimmunity in mouse and human is dysregulation ofFcγRIIb expression resulting in lower surface level of this inhibitoryreceptor, leading to an elevated level of B cell activation andconsequential failure of self-reactive B cell inhibition and productionof plasma cells secreting self-antigen specific immunoglobulins. Suchself-reactive immunoglobulin immune complexes are etiologic agents invarious organ failures in systemic autoimmunity and other arthriticinflammations such as systemic lupus erythematosus (SLE) and rheumatoidarthritis (RA. The immunoglobulins disclosed herein were assessed usinga huPBL-SCID mouse model as a proxy, by examining B cell activitymeasured by the number of B cells and plasma cell development bydetecting the antigen specific immunoglobulins. In this method, humanPBLs from normal or diseased (e.g., SLE or RA) donors are engrafted toimmune-deficient SCID mice and treated with the inhibitoryimmunoglobulin described herein, then challenged with an antigen toexamine the course of B cell development into plasma cells. In suchcase, the production of antigen-specific immunoglobulins is inhibitedfrom which can be inferred inhibition of both B cell activation anddifferentiation.

The protocol for this study is provided in FIG. 11A. Four differentgroups of mice with five mice in each group were engrafted with humanPBLs from a healthy donor. At day 16, test articles consisting of PBS(vehicle control), anti-CD19 with native IgG1 Fc (anti-CD19 IgG1 WT),anti-CD19 with IgG1 Fc of enhanced affinity for FcγRIIb (anti-CD19S267E/L328F) or Rituximab IgG1 anti-CD20 were given 10 mg/kg twiceweekly for a total of 6 doses. At day 24, antigen challenge with tetanustoxoid fragment C was given, and mice were sacrificed at days 31 and 38.Tetanus toxoid (TT) specific antibody production was examined. Theresults of this experiment are shown in FIG. 11B. The data shows thatbefore the antigen challenge, the level of anti-TT specific antibody wasvery low in all the groups. After immunization, the untreated PBScontrol group showed the highest level of anti-TT specific antibodylevel. In comparison, the B cell reducing anti-CD20 antibody producedlow level of antigen specific antibody level. After immunization, theanti-CD19 S267E/L328F group showed the lowest level of antigen specificantibodies, whereas the anti-CD19 IgG1 WT produced a higher level ofantigen specific antibody. These in vivo data show that the anti-CD19antibody with enhanced FcγRIIb affinity is capable of inhibiting B cellactivation and immunoglobulin secreting plasma cell differentiation, andthus support the potential of the immunoglobulins disclosed herein fortreating autoimmune and inflammatory disorders.

Example 6. Co-Engagement of FcγRIIb

The S267E/L328F (high FcγRIIb affinity) variant, along with WT IgG1 andFc-KO variant(s) (̂236R/L328R and/or G236R/L328R) were cloned intoantibodies specific for CD19 expression. FIG. 14A-FIG. 14D list theheavy and light chain variable regions (VH and VL) of the antibodiesused. The VH and VL genes targeting these antigens were constructed bygene synthesis, and variants were constructed, expressed, and purifiedas described above.

The effect of high affinity co-engagement of these antigens with FcγRIIbwas evaluated using the ATP-dependent luminescence B cell viabilityassay as described above. FIG. 12 shows the results. The data furthersupport that CD19 is an effective co-target for using high affinityFcγRIIb co-engagement to inhibit B cell activation. This is consistentwith its role as the signaling component of the BCR complex. Resultsusing two additional anti-CD19 antibodies again confirmed theamenability of this antigen to controlling B cell activation using highaffinity FcγRIIb co-ligation, irrespective of the specific epitopetargeted (FIG. 13).

Example 7. Trial of an Anti-CD19 Antibody S267E/L328F, a ReversibleInhibitor of CD19+ Cells, in IgG4-Related Disease

IgG4-related disease (IgG4-RD) is an immune-mediated conditionresponsible for fibro-inflammatory lesions that can lead to irreversibledamage. No approved therapies for IgG4-RD exist.

A monoclonal anti-CD19 antibody with the Fc modification S267E/L328F wasused to treat IgG4-RD. The anti-CD19 antibody S267E/L328F in thisexample has a heavy chain of SEQ ID NO:9 and a light chain of SEQ IDNO:7, and is a humanized anti-CD19 antibody with an Fc portionengineered for increased affinity (200- to 400-fold over native IgG) toFcγRIIb, the only Fc receptor on B cells. The antibody binds toFcγRIIb+cells and coengages CD19 thereby enhancing the naturalregulatory role of FcγRIIb (inhibition of B cell activation) (FIG. 15B).Co-ligation of CD19 and FcγRIIb leads to downregulation and inhibitionof B lineage cells bearing these targets. Reversible inhibition of Bcell function without B cell ablation is one potential advantage of thisapproach.

The trial was an open-label investigation of the antibody in subjectswith active IgG4-RD having an IgG4-RD Responder Index (IgG4-RD RI) ofand disease activity in one or more organ systems.

The antibody (5 mg/kg) is administered IV every 14 days for up to atotal of 12 infusions. Subjects were included if they had activeIgG4-RD, a compatible pattern of organ involvement consistent withIgG4-RD that cannot be attributed to other causes,histopathologically-proven diagnosis of IgG4-RD, and a peripheral bloodplasmablast count of >900 cells/mL and/or elevated IgG4-RD levels duringscreening.

Fifteen patients were enrolled and received a median number of 7infusions (range 1 to 12). The mean age among the fifteen patientsenrolled was 63 years (range: 43 to 77 years). Ten patients were male,and five were female. Twelve were Caucasian, one was black, and two wereAsian. The patients had a mean serum IgG4 of 220 mg/dL (range: 25-2415;normal 3.9-86.4 mg/dL). The mean baseline IgG4-RD RI score was 12(range: 2-30). The patients had active inflammatory disease in at leastone organ system (range: 1-10, mean 4) (FIG. 16) with a medium of 4organs involved (range 1-10) at the time of study entry. The organs mostcommonly affected were lymph nodes (11 patients; 73%), submandibularglands (9 patients; 60%), parotid glands (8 patients; 53%), and lacrimalglands (7 patients, 47%) (FIG. 17). Organ site involvement occurring ata frequency of 50% included lymph nodes, submandibular glands, andparotid glands. Ten patients (67%) were previously treated. Five of thefifteen patients were on steroids at baseline.

Positron emission tomography (PET) scans were performed at baseline andat three and six months. The primary outcome measure is the proportionof patients on day 169 with decrease in the IgG4-RD RI compared tobaseline. Glucocorticoids are permitted but not required at entry andmust be discontinued by two months. Other immunosuppressive medicationsare not allowed. Mechanistic studies are performed at baseline and atselected intervals.

The study was designed to measure the proportion of patients with animprovement in IgG4-RD activity, wherein an improvement of diseaseactivity is defined by a decrease of IgG4-RD responder index greaterthan or equal to 2 points relative to the pre-administration diseaseactivity score. The study also measured the number and percent ofpatients experiencing a treatment-emergent adverse event as assessed byCTCAE v4.3.

Every other week intravenous administration of the anti-CD19 antibodyS267E/L328F was well tolerated. Fourteen of the fifteen patients dosedwith antibody have showed a decrease of ≧2 points in the IgG4-RD RI,twelve of them within 2 weeks (i.e. after a single dose)(FIG. 18), withresponses deepening over time. Five patients have reached the end of thestudy with an IgG4-RD RI of 0 (no disease activity-definition ofremission). Three patents discontinued treatment prior to the completionof 12 doses. Of the five patients who were on steroids at baseline, allwere able to taper and discontinue.

Administration of the anti-CD19 antibody S267E/L328F demonstrated potentbut reversible B cell inhibition. Following administration of 2 mg/kg ofthe antibody, a rapid drop in cells expressing CD86 was observed, CD86expression then slowly returned to baseline at about 100 days followingadministration of the immunoglobulin (FIG. 19A). CD86 expression is anindication of B cell activation, thus the reduction in CD86 expressionsuggests that the antibody effectively inhibits B cell activation.Further, administration of 2 mg/kg of the antibody resulted in areversible decline in peripheral B cell counts (FIG. 19B). These countsreturned to normal at about 40-60 days following administration of theantibody. Additionally, subjects were challenged with tetanus antigenand then administered varying doses the antibody ranging from 0.03 to 10mg/kg. In subjects administered the antibody, there was a detectabledrop in anti-tetanus IgG relative to placebo-treated subjects (FIG.19C). These results demonstrate that the antibody inhibits B cellactivation in response to antigen challenge.

In subjects with IgG4 related disease administered the antibody, asignificant reduction in both total B cells and plasmablasts was alsoobserved following treatment. Flow cytometry was used to measure countsof total B cells via CD19 and CD79b expression and counts ofplasmablasts via CD38 and CD27 expression. The gating strategy for Bcells and plasmablasts during treatment is shown in FIG. 20.

FIG. 21 shows a summary of the B cell numbers during treatment, bypatient. Total B cell (CD79b+) numbers for eleven of fourteen patientsdecreased quickly to about 40-50% after beginning treatment. Threepatients showed later decreases in total B cell numbers.

FIG. 22A-22B shows a summary of the plasmablasts numbers duringtreatment. Plasmablasts are immature plasma cells. Plasmablasts secretemore antibodies than B cells, but less than plasma cells. They dividerapidly and are still capable of internalizing antigens and presentingthem to T cells. Patients having IgG4-RD have a higher number of CD19+plasmablasts than healthy individuals. FIG. 23 shows that plasmablastsat baseline are elevated (99.07±16.84 CD19+ plasmablasts per 10⁴ CD19+ Bcells (mean±SEM)) as compared to healthy donors (13.73±3.395 CD19+plasmablasts per 10⁴ CD19+ B cells (mean±SEM)). Both CD79b+ plasmablasts(FIG. 22A) and total plasmablasts (FIG. 22A) were reduced 4-5 fold byseven days after the start of treatment in all (fourteen) patients.Notably, plasmablasts decreased by 80-90% in ten of fourteen patients.

Individual B cell and plasmablast counts, as well as serum IgG4 levels(mg/dL) for several patents are discussed further below.

Prior to treatment with the antibody, patient one had an RI of 18 andexhibited IgG4-related sclerosing cholangitis & AIP, tendersubmandibular gland swelling, and tender lacrimal glands. This patientwas previously on steroid therapy. By day 169, this patient had an RI of0, was off steroids, and continued to do well months after the trial.Patient one's B cell and plasmablast counts, as well as serum IgG4levels (mg/dL) is summarized by day of treatment in Table 1 below.

TABLE 1 IgG4 m/dL Day B cells PB (<86) 1 108 1716 349.0 8 55 365 384.015 86 458 354.0 29 67 393 292.5 85 67 397 ND 169 73 603 205.6 197 1221626 222.6

Prior to treatment with the antibody patient two exhibited IgG4-relatedMikulicz disease, orbital disease, lung involvement, and right seminalvesicle involvement. Patient two had an RI of 16 prior to treatment andhad previously been treated with Rituximab. By day 85, this patient hadan RI of 0. Patient two's B cell and plasmablast counts, as well asserum IgG4 levels (mg/dL) is summarized by day of treatment in Table 2below.

TABLE 2 IgG4 m/dL Day B cells PB (<86) 1 76 1247 920 8 70 280 1021 15 77344 969 29 57 188 935 85 60 160 683 169 39 291 513 197 59 522 494

Overall, this data demonstrates that anti-CD19 antibody S267E/L328Ftreatment in active IgG4-RD is tolerated well. Treatment responses(decrease of IgG4-RD RI of 2) observed in 9 of 11 patients (82%).Initial clinical and peripheral blood response to therapy was observedwithin two weeks. Across the patient population sustained improvementwas common. B cell counts fell swiftly by approximately 50%, withplasmablasts falling even more swiftly than B cells. This data suggeststhat the antibody is a rapid and effective agent at treating IgG4-RD.

CD4+ T cells with cytotoxic activity (CD4+CTLs) populations were alsoexamined in the patients receiving anti-CD19 antibody S267E/L328F.CD4+SLAMF7+ CTL numbers have been reported to be increased in theperipheral blood of IgG4-RD patients. Further, circulating CD4+SLAMF7+CTL numbers have been reported to diminish in patients with IgG4-RDafter rituximab therapy. As shown in FIG. 29, CD4+SLAMF7+ CTL numberswere increased in the peripheral blood of IgG4-RD patients (n=101)compared to the controls (n=35). Flow cytometry was used to measurecounts of CD4+CTLs via CD4 and SLAMF7 expression in IgG4-RD patientsreceiving anti-CD19 antibody S267E/L328F. The gating strategy for Bcells and plasmablasts during treatment is shown in FIG. 24. Half of thepatients studied had a high enough percentage of distinct circulatingCD4+ CTL population (10.1%-43.6%) prior to therapy to allow analysisduring the study. Of these, the results show decreases in CD4 CTLs andeffector CD4 CTLs in most of the patients (FIGS. 25A-25B).

B cell apoptosis was also examined in the patients receiving anti-CD19antibody S267E/L328F. Flow cytometry was used to measure counts of Bcells, CD4+ T cells, and CD8+ T cells after therapy. The gating strategyfor B cells, CD4+ T cells, and CD8+ T cells is shown in FIG. 26. Asshown in FIGS. 27A-27L, no significant apoptosis of B cells, CD4+ Tcells, or CD8+ T cells was observed.

BCR linked signaling pathways were also examined in patients afterinfusion of anti-CD19 antibody S267E/L328F. Levels of P-BTK, P-AKT,P-ERK, and P-SYK were examined before treatment (FIG. 28A-28D), 2 hoursafter treatment (FIG. 28E-28H), and 24 hours after treatment (FIG.28I-28L). FIGS. 28A-28L show that BCR induced phosphorylation of Syk andBtk were abolished in CD20+ B cells 2 hours after infusion of theantibody. Downstream Akt signaling pathway was totally blocked in CD20+B cells 2 hours after infusion. Downstream Erk signaling pathway doesnot change after 2 hours, but is partly blocked in CD20+ B cells 24hours after infusion.

Example 8. Dosage Forms of an Anti-CD19 Antibody with the FcModification S267E/L328F

An anti-CD19 antibody with the Fc modification S267E/L328F was preparedin two dosage forms. The first dosage form was a liquid for intravenousinfusion following dilution into normal saline. Each single-use vialcontained 100 mg in 10 mL of phosphate buffered saline. The second formwas a liquid for subcutaneous delivery provided in a single-use vialcontaining 125 mg in 1 mL acetate buffered Proline solution. Thecomposition of the IV formulated pharmaceutical product is provided inTable 3. The composition of the SC formulated pharmaceutical product isprovided in Table 4.

TABLE 3 Anti-CD19 antibody S267E/L328F IV formulation Quantity Name ofIngredient Function Reference (per mL) Anti-CD19 antibody S267E/L328FActive 10.0 mg Sodium Phosphate, Monobasic Buffer USP 0.58 mgMonohydrate Sodium Phosphate, Dibasic Buffer USP 1.55 mg HeptahydrateSodium chloride Tonicity USP 8.77 mg Modifier Water for InjectionSolvent USP qs to 1.0 mL Polysorbate-20 Surfactant USP  0.1 mg

TABLE 4 Anti-CD19 antibody S267E/L328F SC formulation Quantity Name ofIngredient Function Reference (per mL) Anti-CD19 antibody Active  125 mgS267E/L328F Sodium Acetate, Buffer MC 2.35 mg Trihydrate Acetic AcidTonicity Modifier MC 0.16 μL L-Proline Tonicity Modifier MC   30 mgWater for Injection Solvent MC qs to 1.0 mL Polysorbate-80 Surfactant MC 0.1 mg

Example 9. Bioavailability

To characterize and compare the pharmacokinetics (PK) andbioavailability, the anti-CD19 antibody with the Fc modificationS267E/L328F was administered subcutaneously (SC). Study subjects werebetween the ages of 18 and 55 and divided into 3 cohorts. The SCformulations are described in Example 8. Cohort 1 received 125 mg (1 mL)antibody given SC Q14 days×3. Cohort 2 received 50 mg (2 mL) antibodygiven SC Q14 days×3. Cohort 3 received 375 mg (3 mL) antibody given SCQ14 days×3.

Multiple dose SC administration of the antibody was safe and welltolerated at doses of 125 to 375 mg in all 40 subjects administered SCXmAb5871. Treatment emergent adverse events (TEAEs) occurring insubjects receiving any dose of SC XmAb5871 were mild in severity. Theonly drug-related TEAE occurring in more than two subjects who receivedany dose of SC antibody was injection site bruising (three subjects,8%). No subject receiving SC antibody discontinued the study due to anadverse event and there were no serious adverse events during the study.Pharmacokinetic and bioavailability data from the study support an everyother week dosing schedule.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments have been described above for purposes ofillustration, it will be appreciated by those skilled in the art thatnumerous variations of the details may be made without departing fromthe disclosure as described in the appended claims.

1. A method of treating IgG4-related disease (IgG4-RD) in a subject, themethod comprising administering an immunoglobulin that binds FcγRIIb andCD19 on the surface of a B cell, wherein said immunoglobulin comprisesan Fc region, wherein said Fc region is an Fc variant of a parent Fcpolypeptide, comprising at least one amino acid substitution selectedfrom the group consisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N,239D, 267D, 267E, 268E, 268D, 328F, and 328Y, wherein numbering isaccording to the EU index as in Kabat.
 2. A method of reducing at leastone symptom associated with IgG4-RD in a subject, the method comprisingadministering an immunoglobulin that binds FcγRIIb and CD19 on thesurface of a B cell, wherein said immunoglobulin comprises an Fc region,wherein said Fc region is an Fc variant of a parent Fc polypeptide,comprising at least one amino acid substitution selected from the groupconsisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D,267E, 268E, 268D, 328F, and 328Y, wherein numbering is according to theEU index as in Kabat.
 3. The method of claim 2, wherein at least onesymptom associated with IgG4-RD is reduced within 7 days ofadministration of the immunoglobulin.
 4. The method of claim 2, whereinat least one symptom associated with IgG4-RD is reduced within 14 daysof administration of the immunoglobulin.
 5. The method of claim 2,wherein the at least one symptom is exhibited in an organ selected fromlymph nodes, submandibular glands, parotid glands, lacrimal glands,kidney, heart, pericardium, orbit, nasal cavity, lungs, bile ducts,salivary glands, and pancreas.
 6. A method of depleting plasmablasts ina subject with an IgG4-related disease, the method comprisingadministering an immunoglobulin that binds FcγRIIb and CD19 on thesurface of a B cell, wherein said immunoglobulin comprises an Fc region,wherein said Fc region is an Fc variant of a parent Fc polypeptide,comprising at least one amino acid substitution selected from the groupconsisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D,267E, 268E, 268D, 328F, and 328Y, wherein numbering is according to theEU index as in Kabat.
 7. The method of any one of claim 6, wherein thedepletion of plasmablasts is observed within 7 days followingadministration the immunoglobulin that binds FcγRIIb and CD19 on thesurface of a B cell.
 8. The method of claim 6, wherein the plasmablastsare depleted by at least 10% relative the number of plasmablasts priorto the administration of immunoglobulin.
 9. The method claim 6, whereinthe plasmablasts are depleted by at least 20% relative the number ofplasmablasts prior to the administration of immunoglobulin.
 10. Themethod of claim 6, wherein the plasmablasts are depleted by at least 30%relative to baseline.
 11. The method of claim 6, wherein theplasmablasts are depleted by at least 40% relative the number ofplasmablasts prior to the administration of immunoglobulin.
 12. Themethod of claim 6, wherein the plasmablasts are depleted by at least 80%relative the number of plasmablasts prior to the administration ofimmunoglobulin.
 13. A method of reducing CD4+SLAMF7+CTL cell number in asubject with an IgG4-related disease, the method comprisingadministering an immunoglobulin that binds FcγRIIb and CD19 on thesurface of a B cell, wherein said immunoglobulin comprises an Fc region,wherein said Fc region is an Fc variant of a parent Fc polypeptide,comprising at least one amino acid substitution selected from the groupconsisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D,267E, 268E, 268D, 328F, and 328Y, wherein numbering is according to theEU index as in Kabat.
 14. The method of claim 13, wherein the reductionof CD4+SLAMF7+CTL cell number is observed within 24 days followingadministration the immunoglobulin that binds FcγRIIb and CD19 on thesurface of a B cell.
 15. The method of claim 13, wherein theCD4+SLAMF7+CTL cells are reduced by at least 10% relative the number ofCD4+SLAMF7+CTL cells prior to the administration of immunoglobulin. 16.A method of treating a disease in a subject, the method comprisingadministering an immunoglobulin that binds FcγRIIb and CD19 on thesurface of a B cell, wherein said immunoglobulin comprises an Fc region,wherein said Fc region is an Fc variant of a parent Fc polypeptide,comprising at least one amino acid substitution selected from the groupconsisting of 234W, 235I, 235Y, 235R, 235D, 236D, 236N, 239D, 267D,267E, 268E, 268D, 328F, and 328Y, wherein numbering is according to theEU index as in Kabat, and wherein the disease is selected from the groupconsisting of IgG4-related sialadenitis (chronic sclerosingsialadenitis, Küttner's tumour, Mikulicz's disease), IgG4-relateddacryoadenitis (Mikulicz's disease), IgG4-related ophthalmic disease(idiopathic orbital inflammatory disease, orbital pseudotumor), chronicsinusitis, eosinophilic angiocentric fibrosis, IgG4-related hypophysitis(IgG4-related panhypophysitis, IgG4-related adenohypophysitis,gG4-related infundibuloneurohypophysitis, autoimmune hypophysitis),IgG4-related pachymeningitis, IgG4-related leptomeningitis (idiopathichypertrophic pachymeningitis), IgG4-related pancreatitis (Type 1autoimmune pancreatitis, IgG4-related AIP, lymphoplasmacytic sclerosingpancreatitis, chronic pancreatitis with diffuse irregular narrowing ofthe main pancreatic duct), IgG4-related lung disease (Pulmonaryinflammatory pseudotumour), IgG4-related pleuritis, IgG4-relatedhepatopathy, IgG4-related sclerosing cholangitis, IgG4-relatedcholecystitis, IgG4-related aortitis (inflammatory aortic aneurysm),IgG4-related periaortitis (chronic periaortitis), IgG4-relatedperiarteritis, IgG4-related pericarditis, IgG4-related mediastinitis(fibrosing mediastinitis), IgG4-related retroperitoneal fibrosis(retroperitoneal fibrosis, Albarran-Ormond syndrome, Ormond's disease(tetroperitoneal fibrosis)), perirenal fasciitis, Gerota'sfasciitis/syndrome, periureteritis fibrosa, sclerosing lipogranuloma,sclerosing retroperitoneal granuloma, non-specific retroperitonealinflammation, sclerosing retroperitonitis, retroperitoneal vasculitiswith perivascular fibrosis), IgG4-related mesenteritis (subtypes are:mesenteric panniculitis, mesenteric lipodystrophy and retractilemesenteritis) (sclerosing mesenteritis, systemic nodular panniculitis,liposclerosis mesenteritis, mesenteric Weber-Christian disease,mesenteric lipogranuloma, xanthogranulomatous mesenteritis),IgG4-related mastitis (sclerosing mastitis), IgG4-related kidney disease(IgG4-RKD), IgG4-related tubulointerstitial nephritis (IgG4-TIN),IgG4-related membranous glomerulonephritis (idiopathictubulointerstitial nephritis), IgG4-related prostatitis, IgG4-relatedperivasal fibrosis (chronic orchialgia), IgG4-related paratesticularpseudotumor, IgG4-related epididymo-orchitis (paratesticular fibrouspseudotumor, inflammatory pseudotumor of the spermatic cord,pseudosarcomatous myofibroblastic proliferations of the spermatic cord,proliferative funiculitis, chronic proliferative periorchitis,fibromatous periorchitis, nodular periorchitis, reactive periorchitis,fibrous mesothelioma), IgG4-related lymphadenopathy, IgG4-related skindisease (angiolymphoid hyperplasia with eosinophilia, cutaneouspseudolymphoma), IgG4-related perineural disease, and IgG4-relatedthyroid disease (Reidel's thyroiditis), eosinophilic angiocentricfibrosis (affecting the orbits and upper respiratory tract),inflammatory pseudotumour, and multifocal fibrosclerosis.
 17. The methodof claim 16, wherein the disease is selected from the group consistingof autoimmune pancreatitis (lymphoplasmacytic scleorising pancreatitis),eosinophilic angiocentric fibrosis (affecting the orbits and upperrespiratory tract), fibrosing mediastinitis, idiopathic hypertrophicpachymeningitis, idiopathic tubulointerstitial nephritis, inflammatorypseudotumour, Küttner's tumour, Mikulicz's disease, fibrosclerosis,periaortitis, periarteritis, inflammatory aortic multifocal aneurysm,Ormond's disease (tetroperitoneal fibrosis), Riedel's thyroiditis, andsclerosing mesenteritis.
 18. The method of claim 1, wherein said atleast one substitution is selected from the group consisting of 267E and328F.
 19. The method of claim 1, wherein said Fc variant comprises atleast two amino acid substitutions in the Fc region compared to theparent Fc polypeptide, wherein said at least two substitutions isselected from the group consisting of 235D/267E, 235Y/267E, 235D/S267D,235I/267E, 235I/267D, 235Y/267D, 236D/267E, 236D/267D, 267E/328F,267D/328F, 268D/267E, 268D/267D, 268E/267E, and 268E/267D.
 20. Themethod of claim 19, wherein said at least two substitutions are267E/328F.
 21. The method of claim 1, wherein within 2 weeks followingadministration of the immunoglobulin, the subject's IgG4-RD responderindex score (IgG4-RD RI score) is reduced by at least 1 from thebaseline score.
 22. The method of claim 21, wherein the IgG4-RD RI scoreis reduced by 3 within 2 weeks following administration of theimmunoglobulin.
 23. The method of claim 1, wherein the B cell isselected from the group consisting of a plasma cell and a plasmablast.24. The method of claim 1, wherein the B cell is a plasmablast.
 25. Themethod of claim 1, wherein the Fc region that binds FcγRIIb comprisesSEQ ID NO:7 and SEQ ID NO:9.
 26. The method of claim 1, furthercomprising administering standard treatments for an IgG4-related diseaseincluding anti-inflammatory pain reliever drugs (NSAIDs such as aspirin,ibuprofen, naproxen, or Celebrex), acetaminophen, steroids,glucocorticoids (i.e. prednisone), immunosuppressive agents (i.e.azathioprine, mycophenolate mofetil), and immunosuppressive biologics(i.e. rituximab, bortezomib).
 27. The method of claim 1, wherein thesubject is relapsed or refractory to rituximab.
 28. The method of claim1, further comprising tapering and/or discontinuing the use of steroids.29. The method of claim 1, wherein about 1 to about 10 mg/kg body weightof the immunoglobulin is administered to the subject.
 30. The method ofclaim 1, wherein about 5 mg/kg body weight of the immunoglobulin isadministered to the subject.
 31. The method of claim 29, wherein theimmunoglobulin is administered to the subject every 14 days for at least2 doses.
 32. The method of claim 29, wherein the immunoglobulin isadministered to the subject every 14 days for at least 6 doses.
 33. Themethod of claim 29, wherein the immunoglobulin is administered to thesubject every 14 days for at least 12 doses.